This study was supported by the Okayama Spine Group.

This study was approved by the ethics committee at Okayama Rosai Hospital (No. 201-3).
1. Patient examination
<ol>
	<li>History taking
	<ol>
		<li>Evaluate a patient with a suspected herniated disc or stenosis by taking their history. Usually, the patient presents with a history of prodromal low back pain. The patient may correlate their symptoms with an episode of trauma.</li>
		<li>Ask the patient to describe the radiating leg pain, its location, and aggravating and relieving activities.</li>
	</ol>
	</li>
	<li>Physical examination
	<ol>
		<li>To determine the level of the affected nerve, look for signs of motor weakness or loss of sensation in the leg. Check the lumbar range of motion, the straight leg raise (SLR) test, deep tendon reflexes, and muscle weakness.</li>
		<li>Perform the Kemp test to check the location of the herniated discs. If the Kemp test yields a positive result, local pain suggests a facet pathology, while radiating pain into the leg is more suggestive of nerve root irritation, which frequently accompanies foraminal disc herniation or stenosis. 
		NOTE: Usually, the SLR test is negative for patients with lumbar canal stenosis and positive for patients with lumbar disc herniation.</li>
	</ol>
	</li>
</ol>
2. Evaluation of images
<ol>
	<li>Magnetic resonance imaging (MRI)
	<ol>
		<li>Perform an MRI. This is the most accurate diagnostic modality for assessing lumbar disc herniation, showing the site of herniation and the nerve roots affected. The L5 nerve root can be compressed by fibrocartilage at the pars interarticularis of L5.</li>
	</ol>
	</li>
	<li>Computed tomography (CT)
	<ol>
		<li>Use CT to check whether the herniated disc is calcified and to rule out nerve compression by any osteophyte.</li>
	</ol>
	</li>
	<li>CT-MRI fusion imaging
	<ol>
		<li>Use the fusion image to evaluate the feasibility of performing OLIF51. The vascular window, made by the common iliac vessels at the L5-S1 disc level, is clearly visualized. A vascular window of less than 20 mm makes OLIF technically difficult (Figure 1).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63572/63572fig01.jpg" /> 
Figure 1: Preoperative image. (A) Midsagittal reconstruction CT, (B) T2 weighted midsagittal image, (C) CT MRI fusion image. CT and MRI show grade 2 isthmic spondylolisthesis. The vascular window is 37 mm. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Patient positioning and neuromonitoring
<ol>
	<li>Patient positioning
	<ol>
		<li>Place the patient in a right lateral decubitus position on a carbon table. Use an O-arm to obtain 3D images of the lumbar spine of the patient (Figure 2).</li>
		<li>To protect the neurovascular structures, place an axillary roll underneath the patient&#39;s right axilla.</li>
		<li>Flex the hip and knee to 20&#176; to relax the psoas muscle and the lumbar nerve. Secure the patient to the table with tape.</li>
		<li>Insert a percutaneous reference pin into the left sacroiliac joint.</li>
		<li>Obtain 3D images of the O-arm and transmit them to the navigation system.</li>
	</ol>
	</li>
	<li>Neuromonitoring
	<ol>
		<li>Whenever possible, neuromonitor the patient to prevent neurological complications (Figure 3).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63572/63572fig02.jpg" /> 
Figure 2: Patient positioning. Right lateral decubitus position secured with tape. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63572/63572fig03.jpg" /> 
Figure 3: Neuromonitoring. Neuromonitoring is preferable for this technique. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Intraoperative CT and spinal navigation
<ol>
	<li>Intraoperative CT
	<ol>
		<li>Place a navigation reference frame (RF) percutaneously into the contralateral sacroiliac joint or L5 spinous process, and obtain intraoperative CT images (Figure 4).</li>
	</ol>
	</li>
	<li>Navigation
	<ol>
		<li>The navigation system is necessary for this technique. Transmit the CT 3D reconstructed images to the navigation system (Figure 5).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63572/63572fig04.jpg" /> 
Figure 4: O-arm. One O-arm scan is just 23 s. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63572/63572fig05.jpg" /> 
Figure 5: Navigation system. Every instrument is navigated with this system. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Navigated instrument registration
<ol>
	<li>Register every navigated instrument and perform an accuracy check.</li>
</ol>
6. Incision and muscle dissection
<ol>
	<li>Skin incision
	<ol>
		<li>Mark the skin incision using a navigated pointer. Direct the entry point to the center and parallel to the L5-S1 disc space.</li>
		<li>Make a 4 cm long oblique incision on the skin, 4 cm medial to the anterior superior iliac spine and parallel to the pelvis (Figure 6).</li>
	</ol>
	</li>
	<li>Muscle dissection
	<ol>
		<li>Divide the abdominal muscles and dissect along the line of the skin incision. A monopolar cautery is contraindicated due to the risk of injury to the iliohypogastric and ilioinguinal nerves.</li>
		<li>With a retroperitoneal approach, use both index fingers to dissect the retroperitoneal space, following the internal abdominal wall posteriorly down to the psoas muscle, which can then be visualized (Figure 7). 
		CAUTION: If the iliohypogastric nerve is injured, abdominal herniation may occur.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63572/63572fig06.jpg" /> 
Figure 6: Skin incision. The skin is marked using a navigated pointer, which is directed to the center and parallel to the L5-S1 <a href="https://www.jove.com/files/ftp_upload/63572/63572fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63572/63572fig07.jpg" /> 
Figure 7: Navigated pointer. The white arrow indicates a navigated pointer. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Disc preparation and trialing
<ol>
	<li>Disc preparation
	<ol>
		<li>After exposing the promontorium, identify the left common iliac vessels and retract laterally (upward) using a special self-retaining retractor under illumination. Take great care to preserve the left ureter. The L5/S1 intervertebral disc is exposed (Figure 8).</li>
		<li>Retract both common iliac vessels and the bifurcation with three self-retaining retractors. A blunt dissection of the adventitial layers on the L5/S1 disc is necessary to not injure the sympathetic chain. Retrograde ejaculation may occur in male patients if the sympathetic chain is injured.</li>
		<li>After the center of the disc is marked with a navigated pointer, incise the annulus with a knife, and perform a discectomy using Kerrison rongeurs, pituitary forceps, a navigated Cobb elevator, a navigated shaver (Figure 10), a navigated combo tool (periosteum elevator), and a navigated curved curette (Figure 11).</li>
		<li>Use the navigated curved curette to remove the posterior part of the disc. 
		NOTE: Since all the instruments are navigated, the use of an intraoperative C-arm is not required.</li>
	</ol>
	</li>
	<li>Trialing
	<ol>
		<li>After disc preparation, sequentially distract the disc space using navigated trials. Determine the size of the cage on a preoperative lateral radiogram. For Asian patients, a disc height of 10-14 mm and an angulation of 12&#176;-18&#176; is advisable.</li>
		<li>At this step, perform intraoperative radiography to check the implant position and the size of the cage. If the cage protrudes beyond the disc space, choose another adequately sized cage (Figure 12). 
		CAUTION: The surgeon should check the accuracy of the navigation system before using the navigation instruments, as the reference frame may sometimes be accidentally altered. Unlike the C-arm, the disc space in the navigation image does not open on insertion of a trial with an adequate height.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63572/63572fig08.jpg" /> 
Figure 8: Approach to the promontorium. (A) Navigated pedicle probe, (B) navigation monitor.&#160;A navigated pedicle probe (white arrow) is used to check the L5-S1 disc level.&#160;<a href="https://www.jove.com/files/ftp_upload/63572/63572fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63572/63572fig09.jpg" /> 
Figure 9: Self-retaining retractors. These three retractors retract both common iliac vessels and the bifurcation. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/63572/63572fig10.jpg" /> 
Figure 10: Navigated shaving. (A) Navigated shaver, (B) navigation monitor. Navigated shavers are from 6 mm to 12 mm. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63572/63572fig11.jpg" /> 
Figure 11: Navigated curved curette. (A) Navigated curved curette, (B) navigation monitor. A navigated curved curette is very useful to completely remove the posterior part of the disc. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/63572/63572fig12.jpg" /> 
Figure 12: Trialing. After disc preparation, the disc space is sequentially distracted with navigated trials (white arrow). <a href="https://www.jove.com/files/ftp_upload/63572/63572fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
8. Cage placement and screwing
<ol>
	<li>Cage placement
	<ol>
		<li>Insert a mixture of autogenous bone and demineralized bone material into the cage hole. Confirm the angle of insertion for the cage with a navigation pointer to insert the OLIF51 cage accurately.</li>
		<li>Use a mallet to gently insert the OLIF51 cage. After the implant is positioned, take anteroposterior and lateral radiographs to confirm the position of the cage (Figure 13A).</li>
	</ol>
	</li>
	<li>Screwing
	<ol>
		<li>After cage insertion, insert one or two supplemental screws to prevent cage back-out (Figure 13B).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/63572/63572fig13.jpg" /> 
Figure 13: Cage placement and screwing. (A) OLIF51 cage at the L5-S1 disc, (B) cage with screw. A mixture of autogenous bone and demineralized bone material is inserted into the cage hole. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
9. Simultaneous percutaneous pedicle screw (PPS) fixation
<ol>
	<li>Have another surgeon insert the PPSs in the same lateral decubitus position of the patient under navigation guidance.</li>
	<li>Make the screw hole with a navigated high-speed burr, navigated pedicle probe, and navigated tap. Measure the length and diameter of the screws by navigation.</li>
	<li>Insert the S1 PPS in a transdiscal manner to enhance the screw pullout strength.</li>
	<li>Take radiograms after the insertion of the screws (Figure 14).</li>
</ol>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/63572/63572fig14.jpg" /> 
Figure 14: Simultaneous PPS fixation. PPSs are inserted in the same single lateral position by another surgeon under navigation guidance. <a href="https://www.jove.com/files/ftp_upload/63572/63572fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
10. Postoperative procedure
<ol>
	<li>Ambulate the patient 1 day after the surgery. The use of a soft brace is recommended for 3 months. Follow-up at 3 months, 6 months, and 12 months after the surgery to assess for solid bony fusion.</li>
</ol>

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The authors declare that there are no conflicts of interest.

Recently, the lateral lumbar approach for interbody fusion has been gaining popularity due to its minimal invasiveness9. Among these approaches, the direct lateral psoas splitting approach has several disadvantages, such as lumbar nerve plexus injury and psoas muscle weakness10. To reduce these complications, prepsoas or OLIF was introduced by Davis et al. in 201411. However, it is difficult to operate at the L5-S1 disc due to its anatomical features...

Spondylosis is regarded as a stress fracture1 and occurs in about 5% of the young adult population2. The most common level of occurrence is at the L5 level due to the unique shearing force applied at the L5-S1 area. The main symptoms of spondylosis and spondylolisthesis are low back pain, leg pain, and numbness. If conservative treatment proves ineffective, surgical treatment is recommended3. Transforaminal lumbar interbody fusion (TLIF) is an effective and established technique4, but the nonunion rate of this procedure is relatively higher at the L5-S1 level5. Furthermore, with TLIF, it is difficult to create adequate lordosis compared to oblique lumbar interbody fusion (OLIF) or anterior lumbar interbody fusion (ALIF)6.
Indirect decompressions such as ALIF or OLIF are currently common methods for treating lumbar stenosis7. However, the conventional ALIF technique causes a large amount of muscle damage. Oblique lateral interbody fusion at the L5-S1 level (OLIF51) was first reported in 20178. Posterior instrument augmentation is usually necessary to ensure solid fusion, but the conventional OLIF technique uses a C-arm, and the patient position is changed from lateral to prone. To overcome these problems, we report herein a novel technique of C-arm-free simultaneous OLIF51 and percutaneous pedicle screws (PPSs) in a single lateral position. 
We introduce the case of a 75-year-old woman with symptomatic L5 spondylolisthesis (grade 2).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adjustable hinged operating carbon table</td>
			<td>Mizuho OSI</td>
			<td>6988A-PV-ACP</td>
			<td>OSI Axis Jackson table</td>
		</tr>
		<tr>
			<td>CD Horizon Solera Voyager</td>
			<td>Medtronic</td>
			<td>6.4317E+11</td>
			<td>Percutaneous pedicle screw system</td>
		</tr>
		<tr>
			<td>Navigated Cobb elevator</td>
			<td>Medtronic</td>
			<td>NAV2066</td>
			<td></td>
		</tr>
		<tr>
			<td>Navigated combo tool</td>
			<td>Medtronic</td>
			<td>NAV2068</td>
			<td></td>
		</tr>
		<tr>
			<td>Navigated curette</td>
			<td>Medtronic</td>
			<td>NAV2069</td>
			<td></td>
		</tr>
		<tr>
			<td>Navigated high speed bur</td>
			<td>Medtronic</td>
			<td>EM200N</td>
			<td>Stelth</td>
		</tr>
		<tr>
			<td>Navigated passive pointer</td>
			<td>Medtronic</td>
			<td>960-559</td>
			<td></td>
		</tr>
		<tr>
			<td>Navigated pedicle probe</td>
			<td>Medtronic</td>
			<td>9734680</td>
			<td></td>
		</tr>
		<tr>
			<td>Navigated shaver</td>
			<td>Medtronic</td>
			<td>NAV2071</td>
			<td></td>
		</tr>
		<tr>
			<td>NIM Eclipse system</td>
			<td>Medtronic</td>
			<td>ECLC</td>
			<td>Neuromonitouring</td>
		</tr>
		<tr>
			<td>O-arm</td>
			<td>Medtronic</td>
			<td>224ABBZX00042000</td>
			<td>Intraoperative CT</td>
		</tr>
		<tr>
			<td>Radiolucent open spine cramp</td>
			<td>Medtronic</td>
			<td>9731780</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-retaining retractor</td>
			<td>Medtronic</td>
			<td>29B2X10008MDT151</td>
			<td></td>
		</tr>
		<tr>
			<td>Sovereign Spinal System</td>
			<td>Medtronic</td>
			<td>6.4317E+11</td>
			<td>OLIF51 cage</td>
		</tr>
		<tr>
			<td>Spine small passive frame</td>
			<td>Medtronic</td>
			<td>9730605</td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth station navigation system Spine 7R</td>
			<td>Medtronic</td>
			<td>9733990</td>
			<td>Navigation</td>
		</tr>
		<tr>
			<td>U-NavLock Gray</td>
			<td>Medtronic</td>
			<td>9734590</td>
			<td></td>
		</tr>
		<tr>
			<td>U-NavLock Green</td>
			<td>Medtronic</td>
			<td>9734734</td>
			<td></td>
		</tr>
		<tr>
			<td>U-NavLock Orange</td>
			<td>Medtronic</td>
			<td>9734683</td>
			<td></td>
		</tr>
		<tr>
			<td>U-NavLock Violet</td>
			<td>Medtronic</td>
			<td>9734682</td>
			<td></td>
		</tr>
	</tbody>
</table>

c-arm-free simultaneous olif51 and percutaneous pedicle screw fixation in a single lateral position

Oblique lumbar interbody fusion (OLIF) is an established technique for the indirect decompression of lumbar canal stenosis. However, OLIF at the L5-S1 level (OLIF51) is technically difficult because of the anatomical structures. We present a novel simultaneous technique of OLIF51 with percutaneous pedicle screw fixation without fluoroscopy. The patient is placed in a right lateral decubitus position. A percutaneous reference pin is inserted into the right sacroiliac joint. An O-arm scan is performed, and 3D reconstructed images are transmitted to the spinal navigation system. A 4 cm oblique skin incision is made under navigation guidance along the pelvis. The internal/external and transverse abdominal muscles are divided along the muscle fibers, protecting the iliohypogastric and ilioinguinal nerves. Using a retroperitoneal approach, the left common iliac vessels are identified. Special muscle retractors with illumination are used to expose the L5-S1 intervertebral disc. After disc preparation with navigated instruments, the disc space is distracted with navigated trials. Autogenous bone and demineralized bone material are then inserted into the cage hole. The OLIF51 cage is inserted into the disc space with the help of a mallet. Simultaneously, percutaneous pedicle screws are inserted by another surgeon without changing the lateral decubitus position of the patient.
In conclusion, C-arm-free OLIF51 and simultaneous percutaneous pedicle screw fixation are performed in a lateral position under navigation guidance. This novel technique reduces surgical time and radiation hazards.

Fourteen cases (average age: 71.5 years) were treated using this new technique. They were compared to 40 cases (average age: 74.0 years) of L5-S1 TLIF. L5-S1 lordosis angle and disc height were measured in both groups. The OLIF51 group obtained a better L5-S1 lordosis than the TLIF 51 group (Figure 15).
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/63572/63572fig15.jpg" /> 
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Watch this Scientific Journal Video about C-arm-Free Simultaneous OLIF51 and Percutaneous Pedicle Screw Fixation in a Single Lateral Position at JoVE.com

C-arm-Free Simultaneous OLIF51 and Percutaneous Pedicle Screw Fixation in a Single Lateral Position

C-arm-free oblique lumbar interbody fusion at the L5-S1 level (OLIF51) and simultaneous pedicle screw fixation are performed in a lateral position under navigation guidance. This technique does not expose the surgeon or operating staff to radiation hazards.

Oblique lumbar interbody fusion (OLIF) is an established technique for the indirect decompression of lumbar canal stenosis. However, OLIF at the L5-S1 ...

c-arm-free-simultaneous-olif51-percutaneous-pedicle-screw-fixation

Research

JoVE Journal

Medicine

3.8K Views.  Okayama Rosai Hospital. C-arm-free oblique lumbar interbody fusion at the L5-S1 level (OLIF51) and simultaneous pedicle screw fixation are performed in a lateral position under navigation guidance. This technique does not expose the surgeon or operating staff to radiation hazards.

Video: C-arm-Free Simultaneous OLIF51 and Percutaneous Pedicle Screw Fixation in a Single Lateral Position

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression across speech subsystems, and for addressing the critical need for sensitive outcome measures for ongoing experimental treatment trials. To address this need, we are obtaining longitudinal profiles of bulbar impairment in 100 individuals based on a comprehensive instrumentation-based assessment that yield objective measures. Using instrumental approaches to quantify speech-related behaviors is very important in a field that has primarily relied on subjective, auditory-perceptual forms of speech assessment1. Our assessment protocol measures performance across all of the speech subsystems, which include respiratory, phonatory (laryngeal), resonatory (velopharyngeal), and articulatory. The articulatory subsystem is divided into the facial components (jaw and lip), and the tongue. Prior research has suggested that each speech subsystem responds differently to neurological diseases such as ALS. The current protocol is designed to test the performance of each speech subsystem as independently from other subsystems as possible. The speech subsystems are evaluated in the context of more global changes to speech performance. These speech system level variables include speaking rate and intelligibility of speech.
The protocol requires specialized instrumentation, and commercial and custom software. The respiratory, phonatory, and resonatory subsystems are evaluated using pressure-flow (aerodynamic) and acoustic methods. The articulatory subsystem is assessed using 3D motion tracking techniques. The objective measures that are used to quantify bulbar impairment have been well established in the speech literature and show sensitivity to changes in bulbar function with disease progression. The result of the assessment is a comprehensive, across-subsystem performance profile for each participant. The profile, when compared to the same measures obtained from healthy controls, is used for diagnostic purposes. Currently, we are testing the sensitivity and specificity of these measures for diagnosis of ALS and for predicting the rate of disease progression. In the long term, the more refined endophenotype of bulbar ALS derived from this work is expected to strengthen future efforts to identify the genetic loci of ALS and improve diagnostic and treatment specificity of the disease as a whole. The objective assessment that is demonstrated in this video may be used to assess a broad range of speech motor impairments, including those related to stroke, traumatic brain injury, multiple sclerosis, and Parkinson disease.

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis ALS

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...