Name: controlled cervical laceration injury in mice
Uploaded: 2013-05-09T13:00:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about Controlled Cervical Laceration Injury in Mice at JoVE.com

The development of this device was supported by the LISA Co., Louisville, Kentucky. We also acknowledge the ongoing support of Norton Healthcare, Louisville, KY to CBS, and NIH NS050243, NS052290, and NS059622 to XMX.

1. Animal Preparation and Application of the Spine Stabilizer
The mouse cervical spine is concave ventrally as seen from the lateral view. The spinous processes from C3 to T1 are small and friable and, therefore, are not suitable for vertebral stabilization as commonly described 3,4. We recommend that spine stabilization be performed by lateral facet fixation. The fixation device consists of a U-shaped metal channel to support the mouse and two adjustable stainless steel arms that clamp to each facet laterally. This provides excellent immobilization of the target vertebra. After spinal fixation, the spine is slightly elevated to flatten the cervical spine curvature to provide better exposure of the spinal cord.
<ol>
	<li>Sterilize the following surgical instruments: 2-3 pairs of forceps, 2 pairs of microscissors, a 30 G needle, suture and needle holder, skin clips, and clip applicator. Disinfect the spine stabilizer. Anesthetize the mouse using an intraperitoneal cocktail of Ketamine/Xylazine (100 mg / 10 mg/kg). Shave the hair from the mouse's neck.</li>
	<li>After skin cleansing with a povidone-iodine solution and 70% alcohol, move the mouse onto the operating table warmed with a heating pad. Cover the animal's eyes with ophthalmic ointment to prevent corneal drying.</li>
	<li>After induction of anesthesia (reached when the mouse does not respond to a tail pinch), make a posterior cervical midline skin incision from the occiput to the subcutaneous fat-pad of the lower cervical spine. Under magnification, perform a midline incision between the trapezius muscles at C2 and split the semispinalis capitis muscles. Identification of the submuscular fat pad facilitates dissection in the correct layer.</li>
	<li>Extend the midline muscle dissection caudally to the T2 spinous process which serves as a reliable landmark. Cut the muscles attached to the T2 vertebra and remove the cartilaginous portion of the T2 spinous process.</li>
	<li>Dissect the paraspinal muscles from the C2 through the T2 laminae using a pair of micro-scissors. Muscle dissection begins adjacent to the spinous processes and extends bilaterally to the facet joints. Separate the muscles immediately adjacent to the spinous processes and laminae (in the periosteal layer) to minimize bleeding. After the lateral facets are exposed, place the mouse on the U-shaped channel of the LISA stage.</li>
	<li>Attach the stainless steel arms beneath the exposed facets bilaterally. Once the arms are in place, tighten the thumb screws of the steel arms to immobilize the spine. This maintains firm fixation of the target vertebra and provides excellent exposure. The arms can be adjusted to provide precise horizontal orientation of the spine.</li>
	<li>Incise the ligamentum flavum between C5 and C6 to expose underlying dura. Between the interlaminar space, use a 30 G needle to create a small durotomy through which microscissors are placed to extend the durotomy. The spinal cord is now prepared to undergo the controlled laceration lesion.</li>
</ol>
2. Cervical Spinal Cord Laceration Using the LISA Device
<ol>
	<li>The width of the cervical spinal cord enlargement varies at different levels. Make a dorsal hemisection lesion at C5-6 using a 2.3 mm flat blade and set the amplitude of vibration to cover the entire width of the spinal cord. Blades are obtained from Fine Science Tools Inc. (Foster City, CA) and modified for spinal cord laceration. Maintain the amplitude of the blade oscillation at &ge; 0.5 mm, as lower amplitude levels will diminish the ease of cord laceration.</li>
	<li>Position the spine stabilizer and mouse on the LISA stage. The blade is attached to the LISA with its position controlled by micro-drivers capable of three ranges of motion. Components of the LISA and their functions are described in Figure 1.</li>
	<li>Power the blade-vibrating switch on. Under magnification, move the mouse so that the exposed spinal cord is positioned directly beneath the vibrating blade.</li>
	<li>Elevate the stage supporting the mouse towards the oscillating blade. The "0" position is recorded when the blade barely touches the dorsal vein of the spinal cord. Measure the depth of the spinal cord laceration relative to the "0" position.</li>
	<li>Elevate the stage position by micro-driver control: a 360&deg; turn of the micro-driver knob elevates the stage by 0.25 mm. Thus, a 0.75 mm dorsal hemisection lesion is created by turning the micro-driver knob 3 times. The accuracy of the lesion is &plusmn; 0.01 mm. As the blade begins to lacerate the spinal cord, lubricate the surgical field with saline irrigation. The cutting depth of the spinal cord is controlled by the vertical micro-driver and is independent of visual guidance.</li>
	<li>Once the predetermined depth has been reached, turn the vibrating switch off. Ideally, the oscillating blade is positioned in the lesion gap without evidence of tissue deformation. Lower the stage from the cutting blade and remove the blood and saline from the surgical field using cotton Q-tips. Hemostasis occurs spontaneously in &lt; 1 min.</li>
	<li>Release the mouse from the spine stabilizer. Approximate the paraspinal muscles using 6-0 silk suture and close the skin wound using stainless steel Michel clips.</li>
</ol>
3. Animal Care
<ol>
	<li>Subcutaneously inject a total of 1-2 ml saline to maintain adequate hydration and place the mouse in the recovery cage on a heating pad while regaining consciousness.</li>
	<li>Provide water and soft food ad lib and administer analgesics for 48 hours post-operatively. There is no need for bladder care following dorsal hemisection of the spinal cord.</li>
</ol>

<ol>
	<li>Blackmore, M., Letourneau, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+within+maturing+neurons+limit+axonal+regeneration+in+the+developing+spinal+cord.">Changes within maturing neurons limit axonal regeneration in the developing spinal cord.</a> J. Neurobiol. 66 (4), 348 (2006).</li><li>Blackmore, M. G., 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=Kruppel-like+Factor+7+engineered+for+transcriptional+activation+promotes+axon+regeneration+in+the+adult+corticospinal+tract.">Kruppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract.</a> Proc. Natl. Acad. Sci U.S.A. 109 (19), 7517 (2012).</li><li>Carbajal, K. S., 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=Surgical+transplantation+of+mouse+neural+stem+cells+into+the+spinal+cords+of+mice+infected+with+neurotropic+mouse+hepatitis+virus.">Surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus.</a> J. Vis. Exp. (53), e2834 (2011).</li><li>Duhamel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lumbar+and+cervical+spinal+cord+blood+flow+measurements+by+arterial+spin+labeling:+sensitivity+optimization+and+first+application.">Mouse lumbar and cervical spinal cord blood flow measurements by arterial spin labeling: sensitivity optimization and first application.</a> Magn. Reson. Med. 62 (2), 430 (2009).</li><li>Hermanns, S., Reiprich, P., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+method+to+reduce+collagen+scar+formation+in+the+lesioned+rat+spinal+cord.">A reliable method to reduce collagen scar formation in the lesioned rat spinal cord.</a> J. Neurosci. Methods. 110 (1-2), 141 (2001).</li><li>Hill, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomic+and+functional+outcomes+following+a+precise,+graded,+dorsal+laceration+spinal+cord+injury+in+C57BL/6+mice.">Anatomic and functional outcomes following a precise, graded, dorsal laceration spinal cord injury in C57BL/6 mice.</a> J. Neurotrauma. 26 (1), 1 (2009).</li><li>Iannotti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dural+repair+reduces+connective+tissue+scar+invasion+and+cystic+cavity+formation+after+acute+spinal+cord+laceration+injury+in+adult+rats.">Dural repair reduces connective tissue scar invasion and cystic cavity formation after acute spinal cord laceration injury in adult rats.</a> J. Neurotrauma. 23 (6), 853 (2006).</li><li>Inman, D., Guth, L., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influences+on+secondary+degeneration+and+wound+healing+following+spinal+cord+injury+in+various+strains+of+mice.">Genetic influences on secondary degeneration and wound healing following spinal cord injury in various strains of mice.</a> J. Comp Neurol. 451 (3), 225 (2002).</li><li>Onifer, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+forelimb+dysfunction+after+dorsal+cervical+spinal+cord+injury.">Adult rat forelimb dysfunction after dorsal cervical spinal cord injury.</a> Exp. Neurol. 192 (1), 25 (2005).</li><li>Ramer, M. S., Harper, G. P., Bradbury, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+spinal+cord+research+-+a+refined+strategy+for+the+International+Spinal+Research+Trust.">Progress in spinal cord research - a refined strategy for the International Spinal Research Trust.</a> Spinal Cord. 38 (8), 449 (2000).</li><li>Seitz, A., Aglow, E., Heber-Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+from+spinal+cord+injury:+a+new+transection+model+in+the+C57Bl/6+mouse.">Recovery from spinal cord injury: a new transection model in the C57Bl/6 mouse.</a> J. Neurosci. Res. 67 (3), 337 (2002).</li><li>Sivasankaran, R., 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=PKC+mediates+inhibitory+effects+of+myelin+and+chondroitin+sulfate+proteoglycans+on+axonal+regeneration.">PKC mediates inhibitory effects of myelin and chondroitin sulfate proteoglycans on axonal regeneration.</a> Nat. Neurosci. 7 (3), 261 (2004).</li><li>Yu, P., 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=Inhibitor+of+DNA+binding+2+promotes+sensory+axonal+growth+after+SCI.">Inhibitor of DNA binding 2 promotes sensory axonal growth after SCI.</a> Exp. Neurol. 231 (1), 38 (2011).</li><li>Zhang, Y. 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<ol>
		<li>Several authors (YPZ, XMX, CBS) have a financial interest in the Louisville Impactor System, Inc.</li>
	 <li>The authors, Yi Ping Zhang, Lisa B. E. Shields, and Christopher B. Shields, are employees of Norton Healthcare, Louisville, KY. Other authors are employed by Indiana University, Indianapolis, IN.</li>
	 <li>The authors did not receive funding from any company that produce reagents or instruments utilized in this article.</li>
	</ol>

Vertebral stabilization prior to laceration injuries to the spinal cord has been obtained by fixation of the spinous processes. Both the cervical spine lordotic curve and attachment of clamps to the friable short cervical spinous processes from C3 through T1 in the mouse prevent effective spine stabilization. Furthermore, use of a razor blade or microscissors utilized under manual control causes significant tissue deformation that creates variability in the depth of the lesion 6. This may lead to the misinterpretation of data particularly when axonal regeneration of specific pathways is studied. For example, spared dorsal corticospinal axons may be misinterpreted as regenerated axons if the dorsal corticospinal tract was not completely transected at the time of lesioning. These challenges can be overcome by using a spine stabilization device with fixation to the facets at a single level and precise lesioning of the spinal cord. Additionally, using a high frequency oscillating blade produces a sharp laceration without crushing or contusing the adjacent spinal cord. This method has been used to produce spinal cord laceration injuries in rats 9,12,14, with subsequent modifications to produce thoracic spinal cord lacerations in mice 6. In the present communication, we describe the method of creating reliable cervical laceration lesions in the mouse.
Insofar as the anteroposterior diameter of the spinal cord is &lt;2 mm in the mouse, precise depths of the laceration lesion are vital in creating a reliable experimental model. Minimal variability in the lesion depth will significantly alter results of experiments assessing axon regeneration as well as volumetric and behavioral studies. The accuracy of the lesion depth using this method is &plusmn; 0.01 mm because we used high precision micro-drivers to control the position of the cutting blade. This method has reduced the inconsistency inherent in other models of creating a laceration SCI. This method is particularly useful in studying axonal regeneration of the long spinal cord pathways located in the dorsal half of the spinal cord, such as the corticospinal tract, the rubrospinal tract, and the dorsal ascending tract. With this method, these fiber tracts can be completely and reliably transected. In this respect, errors of data interpretation are minimized, thereby improving reliability of reporting of experimental studies on SCI.
In summary, we have described a novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade. Using this method in a dorsal thoracic spinal cord laceration model in mice 6, we demonstrated a tight correlation between the laceration depth, histology, and behavior recovery. Such a technique has also been found to be reliable by several other laboratories 2,12.

The availability of genetically modified mice is a powerful tool to identify the effects of specific genes that play a role in the mechanisms of SCI. Laceration SCI is an important model used to examine therapeutic agents or molecules that may provide effective treatment following this injury 8. Fixation of the spinous processes during creation of the laceration injury in mice is imprecise due to the difficulty in grasping the thin and fragile spinous processes involved with maintaining spinal fixation 5,11. Variability in the depth of the laceration of only 0.2 mm (10% of the diameter of the mouse spinal cord) causes misleading interpretation of data. The nature and extent of the spinal cord laceration lesion must be precisely defined 10. To address this challenge, we have developed a novel technique consisting of vertebral stabilization and used fabricated blades attached to the Louisville Injury System Apparatus (LISA) to produce a laceration SCI 7,14. This injury was created by using a sharp oscillating blade that avoided tissue deformation during the laceration process. The depth of the laceration was precise to an accuracy of 0.01 mm by using micro-drivers which control the laceration depth. Cutting blades are custom-made to specific shapes and widths to create the desired laceration contour 9. We demonstrate 1) the method of cervical spine exposure, 2) the technique of vertebral stabilization using a bilateral facet fixation device, and 3) the creation of a cervical laceration injury using a vibrating blade.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Mice vertebral stabilizer </td>
 <td>Louisville Impactor System</td>
 <td></td>
 <td>Stabilize and expose the cervical vertebra</td>
 </tr>
 <tr>
 <td>LISA vibraknife </td>
 <td>Louisville Impactor System</td>
 <td></td>
 <td>Produce the laceration injury of the cervical spinal cord</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>Fine Science Tools (USA) </td>
 <td>15013-12</td>
 <td>Skin and trapezius muscle incision</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>Fine Science Tools (USA) </td>
 <td>15023-10</td>
 <td>Separate muscles from the laminae</td>
 </tr>
 <tr>
 <td>Spring Scissors</td>
 <td>Fine Science Tools (USA) </td>
 <td>15002-08</td>
 <td>Incision of dura</td>
 </tr>
 <tr>
 <td>Graefe forceps </td>
 <td>Fine Science Tools (USA) </td>
 <td>11154-10</td>
 <td>Retract skin</td>
 </tr>
 <tr>
 <td>Dumont #7 forceps </td>
 <td>Fine Science Tools (USA) </td>
 <td>11274-20</td>
 <td>Muscle retraction (tip modified)(Fig. A)</td>
 </tr>
 <tr>
 <td>Dumont SS forceps </td>
 <td>Fine Science Tools (USA) </td>
 <td>11203-25</td>
 <td>Fixation of vertebra (tip modified )(Fig.B)</td>
 </tr>
 <tr>
 <td>30G needle </td>
 <td>Becton Dickenson</td>
 <td>305106</td>
 <td>Create a dural opening </td>
 </tr>
 <tr>
 <td>6-0 suture </td>
 <td>Ethicon </td>
 <td>8806H</td>
 <td>Close muscle and fascial layers </td>
 </tr>
 <tr>
 <td>wound clip </td>
 <td>Fine Science Tools (USA) </td>
 <td>12031-07</td>
 <td>Skin closure</td>
 </tr>
 <tr>
 <td>Tribromoethanol (Avertin)</td>
 <td>Sigma-Aldrich</td>
 <td>90710-10G</td>
 <td>Anesthetic agent</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Louisville Impactor System, Inc, 210 E. Gray St., Suite 1102, Louisville, KY 40202, (502) 629-5510, E-mail: <a href="mailto:cbshields1@gmail.com">cbshields1@gmail.com</a>
 Fine Science Tools (USA), Inc, 373-G Vintage Park Drive, Foster City, CA 94404-1139, (800) 521-2109, E-mail: <a href="mailto:info@finescience.com">info@finescience.com</a> 
 Becton Dickenson, 1 Becton Drive, Franklin Lakes, NJ USA 07417, (201) 847-6800 Ethicon, Route 22 West,
 Somerville, NJ 08876 1-877-ETHICON 
 Sigma-Aldrich Corp. 
 St. Louis, MO, USA, 63178 (314) 771-5765, E-mail: cssorders@sial.com</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Figure A is the modified Dumont #7 forceps; B is the modified Dumont SS forceps.</td>
 </tr>
 </tbody>
 </table>

controlled cervical laceration injury in mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

Immobilization of the target vertebra is of great importance in generating precise lesions of the mouse spinal cord. Our spine stabilization device overcomes the anatomical issues of short spinous processes and ventral lordosis of the mouse cervical spine. The cervical vertebrae are well exposed using our cervical spine stabilizer (Figure 2). Our mouse spine stabilizing device is a reliable technique to prepare the spine for cervical spinal cord procedures. The depth of the lesion using the LISA is accurate to 0.01 mm 6,13. The precise laceration causes no contusion at the lesion/tissue interface (Figure 3). The precision of the dorsal hemisection lesions was demonstrated in C57BL/6 mice in a study on axonal regeneration in which a 0.9 mm deep laceration extended just beyond the central canal in each specimen confirmed by pathological sections of the spinal cord 1. Locomotion of all of these animals recovered following this spinal cord laceration injury.
<img alt="Figure 1" src="/files/ftp_upload/50030/50030fig1.jpg" /> 
Figure 1. (A) The mouse in the spine stabilizer placed on the LISA stage. The vibrating blade is directed toward the spinal cord to be lacerated. Micro-driver controls are located beneath the stage and are designed to position the mouse in the appropriate site. The vertical micro-driver controls the lesion depth, and the tilting knob controls the horizontal plane of the spinal cord to prevent angulation of the laceration. The on-off switch controls the vibration motor, and another knob adjusts its amplitude. (B) A 0.75 mm dorsal hemisection laceration lesion cut beneath intact laminar arches.
<img alt="Figure 2" src="/files/ftp_upload/50030/50030fig2.jpg" /> 
Figure 2. (A) The mouse spine stabilizer consisting of a U-shaped channel and two arms and connectors. The mouse is placed in the C trough used for cervical SCI and in the T trough for thoracic SCI. (B) The cervical spine is fixated by placing the arms under the lateral facets and then locking the thumb screws. The dura is exposed between the laminae of C5-6, C6-7, and C7-T1 without any removal of bone.
<img alt="Figure 3" src="/files/ftp_upload/50030/50030fig3.jpg" /> 
Figure 3. Four dorsal spinal cord lacerations at depths of 0.5, 0.8, 1.1, and 1.4 mm observed in the sagittal view (cresyl-violet and eosin stain) depicting the high degree of precision using this technique.

Watch this Scientific Journal Video about Controlled Cervical Laceration Injury in Mice at JoVE.com

Controlled Cervical Laceration Injury in Mice

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

controlled-cervical-laceration-injury-in-mice

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Medicine

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...