Name: Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey (Video) | JoVE Journal
Uploaded: 2014-10-14T13:00:00.000+00:00
Duration: 8 min 11 s
Description: Learn complete spinal cord transection and brain dissection techniques in larval sea lamprey for studying axon guidance after injury. This method enables wholemount visualization of regenerating spinal-projecting neurons.

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5 minutes

Supported by NIH Grants NS14837, R01 NS38537, R24 HD050838 to Dr. Michael E. Selzer; Shriners Research Grant SHC-85220 to Dr. Michael E Selzer; and Shriners Research Grant SHC-85310 to Dr. Michael I. Shifman. Dr. Ant&#243;n Barreiro-Iglesias was supported by the Fundaci&#243;n Barri&#233; (Spain) and the Xunta de Galicia (Galicia, Spain).

See Table 1 for all materials used in this protocol.
Experiments were approved by the Institutional Animal Care and Use Committee at Temple University.
1. Animals
<ol>
	<li>Obtain wildtype larval sea lampreys (Petromyzon marinus L.), 10 &#8211; 14 cm in length (4 &#8211; 7 years old) from streams feeding Lake Michigan, from tributaries to the Delaware River (Pennsylvania) or streams in Maine.</li>
	<li>In the laboratory, maintain larvae in groups of 50 - 100 animals in 50 gallon freshwater tanks at 16 &#176;C. Line the tanks with an inch of gravel for the larvae to burrow in, and the water is charcoal filtered and conditioned with air stones for at least 48 hr to remove chlorine and other impurities before placing the lampreys in them. There is no need to feed the animals, which recycle their own detritus, up to the day of use.</li>
</ol>
2. Complete Spinal Cord Transection
<ol>
	<li>Before the spinal cord surgery prepare a lamprey Ringer solution with the following composition: 110 mM NaCl, 2.1 mM KCl, 2.6 mM CaCl2, 1.8 mM MgCl2 and 10 mM Tris buffer; pH 7.4.</li>
	<li>To perform the spinal cord transection, place the larvae in a small container to be anesthetized with 1% tricaine methanesulfonate in ice cold Ringer solution.</li>
	<li>Once anesthetized place the animals in a petri dish half filled with Sylgard (silicone) and use 4 insect pins (0.15 mm diameter) to hold the animals in position for the surgery (one is placed at the snout of the animal, one at the tail and two at the level of the 5th gill). Fill the petri dish almost up to the top with Ringer solution. Put the dish with the larva on ice while performing the surgery under the stereomicroscope.</li>
	<li>To perform the spinal cord transection a dorsal, midline, make a longitudinal incision with a scalpel (#11) at the level of the 5th gill to visualize the spinal cord. Initially the skin is open by inserting the tip of the scalpel blade facing up and then the muscle tissue is cut very gently until the spinal cord can be seen from above. Fashion two hooks from the insect pins and use them to hold the body walls open while performing the spinal cord transection.</li>
	<li>Completely transect the spinal cord at the level of the 5th gill by using a pair of scissors (Castroviejo #8), insert the scissors perpendicularly to the spinal cord. The spinal cord has to be completely transected with one clean cut. Visualize the spinal cord cut ends under the stereomicroscope to confirm that the spinal cord has been completely transected. Then, realign the cut ends of the spinal cord after the transection by using a pair of forceps (#5).</li>
	<li>After performing the spinal cord transection close the wound in the dorsal body wall with a pair of fine forceps and put the larvae on ice for 1 hr to allow the wound to dry. Put a piece of filter paper between the ice and the larvae and soak it with lamprey Ringer solution. The low temperature keeps the animals alive at the same time that it allows the wound to air dry and therefore helping to prevent infections.</li>
	<li>Then transfer the larvae to small tanks (1 larva per tank) with ice cold water for 24 hr.</li>
	<li>Examine the lesioned larvae 1 day after the spinal transection to behaviorally confirm that there is no movement caudal to the site of injury. A spinal cord transection is considered complete if the larva can move only its head rostral to the site of injury (at the level of the 5th gill). If this is the case the larvae can now be transferred to tanks (1 larva per tank) with freshwater at room temperature (23 &#176;C) to allow them to recover for the chosen time points.</li>
	<li>During the recovery period change the water every 2 to 3 days.</li>
</ol>
3. Brain Dissection and Preparation of the Brain for in situ Hybridization
<ol>
	<li>At the desired time points after the injury re-anesthetize the larval sea lampreys and place and pin them in the petri dish with Ringer solution to perform the brain dissection as above.</li>
	<li>To dissect the brain out, first make a transverse incision with the scalpel between the nostril and the pineal organ. From here begin to cut the tissue surrounding the brain by using a pair of Castroviejo scissors while holding it with a pair of forceps.</li>
	<li>Once the brain is exposed, remove the choroid plexus by using a pair of forceps and use the hooks to spread the head of the animal and expose the cranial nerves. Then, cut the spinal cord transversally with the Castroviejo scissors. Hold the spinal cord with a pair of forceps while using another pair of forceps to cut the cranial nerves to dissect the brain out.</li>
	<li>After dissection, put the brain on a small piece of silicone and hold it in position by inserting 2 insect pins in the olfactory bulbs and 1 in the spinal cord. Then, cut the posterior and cerebrotectal commissures of the brain along the dorsal midline with the scissors and deflect the alar plates laterally and pin them flat to the silicone with 4 insect pins (Figure 2).</li>
	<li>Transfer the piece of silicone with the brain to a tube containing 4% paraformaldehyde in phosphate buffered saline (pH 7.4) to be fixed at room temparature for 3 hr on a rotator.</li>
	<li>After fixation use the brain for wholemount in situ hybridization as previously described (Swain et al., 1994). Brains from control or sham operated animals should be run always in parallel with the brains from injured animals. The wholemounted larval brain can be studied by in situ hybridization because of its flat and thin shape and also due to the absence of myelin10, which makes the brain fairly translucent.</li>
</ol>

<ol><li>Rodicio, M. C., Barreiro-Iglesias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lampreys+as+an+animal+model+in+regeneration+studies+after+spinal+cord+injury%5BTitle%5D)+AND+%22Rev+Neurol%22%5BJournal%5D)">Lampreys as an animal model in regeneration studies after spinal cord injury.</a> Rev Neurol. 55, 157-166 (2012).</li>
<li>Davis, G. R. Jr, McClellan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extent+and+time+course+of+restoration+of+descending+brainstem+projections+in+spinalcord-transected+lamprey%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Extent and time course of restoration of descending brainstem projections in spinalcord-transected lamprey.</a> J Comp Neurol. 344, 65-82 (1994).</li>
<li>Jacobs, A. J., Swain, G. P., Snedeker, J. A., Pijak, D. S., Gladstone, L. J., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recovery+of+neurofilament+expression+selectively+in+regenerating+reticulospinal+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.</a> J Neurosci. 17, 5206-5220 (1997).</li>
<li>Shifman, M. I., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+netrin+receptor+UNC-5+in+lamprey+brain+modulation+by+spinal+cord+transection%5BTitle%5D)+AND+%22Neurorehabil+Neural+Repair%22%5BJournal%5D)">Expression of the netrin receptor UNC-5 in lamprey brain modulation by spinal cord transection.</a> Neurorehabil Neural Repair. 14, 49-58 (2000).</li>
<li>Barreiro-Iglesias, A., Laramore, C., Shifman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+sea+lamprey+UNC5+receptors+cDNA+cloning%2C+phylogenetic+analysis+and+expression+in+reticulospinal+neurons+at+larval+and+adult+stages+of+development%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">The sea lamprey UNC5 receptors cDNA cloning, phylogenetic analysis and expression in reticulospinal neurons at larval and adult stages of development.</a> J Comp Neurol. 520, 4141-4156 (2012).</li>
<li>Shifman, M. I., Yumu, lR. E., Laramore, C., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+repulsive+guidance+molecule+RGM+and+its+receptor+neogenin+after+spinal+cord+injury+in+sea+lamprey%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Expression of the repulsive guidance molecule RGM and its receptor neogenin after spinal cord injury in sea lamprey.</a> Exp Neurol. 217, 242-251 (2009).</li>
<li>Busch, D. J., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synuclein+accumulation+is+associated+with+cell-specific+neuronal+death+after+spinal+cord+injury%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Synuclein accumulation is associated with cell-specific neuronal death after spinal cord injury.</a> J Comp Neurol. 520, 1751-1771 (2012).</li>
<li>Shifman, M. I., Zhang, G., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delayed+death+of+identified+reticulospinal+neurons+after+spinal+cord+injury+in+lampreys%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.</a> J Comp Neurol. 510, 269-282 (2008).</li>
<li>Swain, G. P., Jacobs, A. J., Frei, E., Selzer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+in+situ+hybridization+in+wholemounted+lamprey+brain+neurofilament+expression+in+larvae+and+adults%5BTitle%5D)+AND+%22Exp.+Neurol%22%5BJournal%5D)">A method for in situ hybridization in wholemounted lamprey brain neurofilament expression in larvae and adults.</a> Exp. Neurol. 126, 256-269 (1994).</li>
<li>Bullock, T. H., Moore, J. K., Fields, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolution+of+myelin+sheaths%3A+both+lamprey+and+hagfish+lack+myelin%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">Evolution of myelin sheaths: both lamprey and hagfish lack myelin.</a> Neurosci Lett. 48, 145-148 (1984).</li>
<li>Cohen, A. H., Kiemel, T., Pate, V., Blinder, J., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature+can+alter+the+function+outcome+of+spinal+cord+regeneration+in+larval+lampreys%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Temperature can alter the function outcome of spinal cord regeneration in larval lampreys.</a> Neuroscience. 90, 957-965 (1999).</li>
</ol>

The authors declare that they have no competing financial interests.

Here we present a detailed protocol to perform a complete spinal cord transection and posterior brain dissection in larval sea lampreys. This procedure allows analyzing differences in gene expression between identifiable spinal cord projecting neurons after spinal cord injury by means of a whole-mount brain in situ hybridization. The critical step in the procedure is the correct performance of a complete spinal cord transection, which can be controlled by observing the cut ends of the spinal cord under the stereomicrocop...

In mammals spinal cord injury (SCI) is a devastating condition that leads to permanent loss of function below the site of injury because injured axons do not regenerate through the trauma zone and reconnect to their appropriate targets. In contrast to mammals, lampreys recover locomotion after a complete spinal cord injury.1 Interestingly, lampreys have a set of 36 spinal cord projecting neurons that are individually identifiable in whole-mount brain preparations because of their big size2,3 (Figure 1). All of these spinal-projecting neurons are axotomized by a high-level complete spinal cord transection. Previous studies of our group and others have shown that even in the presence of functional recovery after SCI some of these neurons show a very low regenerative capacity (they are considered &#8220;bad regenerators&#8221;), while others usually regenerate their axon through the site of injury (they are considered &#8220;good regenerators&#8221;).2,3 This characteristic makes lampreys an interesting vertebrate model to study the differences in gene expression between good and bad regenerator spinal-projecting neurons that in turn will lead to the differences in the intrinsic regenerative ability of neurons that attempt to regenerate their axons in the same extrinsic environment.1
Using this model we have previously shown that spinal-projecting neurons with low regenerative ability show expression of axonal guidance molecule receptors like UNC54,5 and neogenin,6 which mediate the inhibitory action of netrin and RGM respectively. In addition, by using this method our group has also shown that only the good regenerators show a recovery of the expression of neurofilaments after the injury and during the regeneration process. Recently, Busch and Morgan7 have shown by immunofluorescence that the bad regenerators show an increased expression of synuclein after injury, which has been related by the authors to the fact that the &#8220;bad regenerator&#8221; spinal-projecting neurons slowly die after a complete spinal cord transection5,7,8. So, the lamprey model of a complete spinal cord injury has emerged as a very useful model to understand what makes a spinal cord projecting neuron a &#8220;bad regenerator&#8221; after axotomy.
To conduct our studies we are performing a complete spinal cord transection surgery protocol and a posterior brain dissection at the desired time points after injury to perform wholemount in situ hybridization. In the present methodological article we present a detailed protocol for the proper performance of a complete spinal cord injury surgery in larval lampreys, the subsequent maintenance of the animals and the final brain dissection and preparation of the brain for a wholemount in situ hybridization. A detailed protocol to perform the wholemount in situ hybridization in the brain of larval lampreys has been previously reported.9 In addition, this protocol for spinal cord injury and brain dissection can be also used to then process the brains for immunohistochemistry or other histological methods.

<table><tbody><tr><td>Tricaine methane sulfonate</td><td>Spectrum</td><td>TR108</td><td>Benzocaine saturated solution in PBS for sacrifice</td></tr><tr><td>Scalpel #3</td><td>Fine Science Tools (FST)</td><td>10003-12</td><td></td></tr><tr><td>Blades for scalpel: #11</td><td>Fine Science Tools&amp;nbsp;</td><td>10011-00</td><td></td></tr><tr><td>Castroviejo scissors #8</td><td>Fine Science Tools&amp;nbsp;</td><td>15002-08</td><td></td></tr><tr><td>Forceps #4 &amp;amp; #5</td><td>Dumont, Switzerland</td><td>Roboz RS4955</td><td>#4 for dissection of Spinal cord; #5 for stripping menninges</td></tr><tr><td>Dissecting Microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Sylgard</td><td>Dow Corning Co.</td><td>184</td><td></td></tr><tr><td>Insect pins 0.15, 0.20 mm</td><td>Austerlitz</td><td>No catalogue #</td><td>0.15 mm for pinning brain and spinal cord; 0.20 mm for the body</td></tr><tr><td>7 ml HDPE Scintillation Tubes with Caps</td><td>Fisher Scientific</td><td>03-337-1</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Science (EMS)</td><td>19210</td><td>Dilute to 4% in PBS</td></tr></tbody></table>

complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

As an example of the results that can be obtained when using this method, representative images of wholemounted brains showing the expression of the neogenin transcripts in identifiable spinal cord-projecting neurons of control and 2 weeks post lesion larval sea lampreys are shown in Figure 2. The readers are referred to a previous study6 reporting the relationship between the expression of neogenin after a complete spinal cord transection and the regenerative ability of the identifiable spina...

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

The overall goal of this procedure is to perform a complete spinal cord, transection and brain dissection for the study of Axon guidance after injury. This is accomplished by first transecting, a lampre spinal cord at the level of the fifth gill. Then the brain is dissected and pinned to syl guard.After fixing the brain, it can be stored in methanol or immediately used for in situ hybridization ultimately changes in the expression of axonal. Guidance receptors can be seen in a regenerating vertebrae after a spinal cord injury. The advantage of the technique we're going to demonstrate today over more conventional paraffin sectioning or cryostat sectioning, is that it allows you to see the whole brain, including neurons that project to the spinal cord that are large and identified.Demonstrating the procedure is Dr.Kathy Zang, a research scientist in our laboratory From the streams feeding Lake Michigan tributaries to the Delaware River in Pennsylvania, or streams in Maine. Wild type larval sea lampre of the species. Pet mize on Marus were obtained.Four to 7-year-old larvae are 10 to 14 centimeters in length and large enough for the injury and dissection. Maintain them in groups of 52, a hundred. In 50 gallon fresh water tanks at 16 degrees Celsius.The tank requires a one inch gravel substrate so the lamprey can borrow All water added to the tank should be conditioned with charcoal filtering and aeration for at least 48 hours before it is added to the tank. Provided the animals are used within one year, there is no need to feed them for this procedure. Have freshly prepared ringer solution at the ready.First transfer a lampe to saturated aqueous benzocaine solution. Once anesthetized, transfer it to a dish half filled with sil guard. Put the dish on ice and top the dish off with ice.Cold ringers. Secure the animal with three 0.15 diameter insect pins, one at the snout and two at the fifth gill. Then position it under a stereo microscope for the surgery.Begin by using a number 11 scalpel to make a longitudinal incision along the dorsal midline at the level of the fifth gill. This will reveal the spinal cord while making this incision. Use two hooks fashioned from insect pins to secure the body walls using Castro reveal number eight, scissors.Make a perpendicular cut across the exposed spinal cord. Inspect the cut ends of the spinal cord to ensure that the transection is complete. Then using forceps, realign the cut ends and close the wound.Transfer the lampre lava onto a piece of filter paper soaked in ringer solution. Move the preparation onto a bed of ice for an hour so the wound can dry after an hour. Transfer the lava to its own small tank of ice cold water.Allow it to heal there for 24 hours. After 24 hours, check for movement cordal to the transection. If there are any movements, the transection was incomplete.If there aren't, then the transect was good and the animal can be moved to a room temperature tank for recovery while the animal recovers. Change the water every two to three days. Once the transected animal is sufficiently recovered, anesthetize it and prepare it for dissection as previously described.To dissect the brain, start with a transverse incision between the nostril and the pineal organ. Then with Castro veal, scissors cut the tissue surrounding the brain while securing the animal. With forceps.The white oval structures on either side of the brain stem are the otic capsules. Once the brain is exposed, use forceps to remove the choroid plexus. Next, use the modified pin hooks to spread the head of the animal and expose the cranial nerves.Then cut the spinal cord transversely with the scissors while securing the spinal cord with forceps. Cut the cranial nerves to dissect out the brain. Then transfer the brain to a small piece of silicone and secure it there.Using one insect pin in the spinal cord and one pin through one of the olfactory bulbs. Then using the scissors, cut the inter olfactory bulb commissure the cerebral protector commissure and the oex of the brainstem along the dorsal midline. Once the commissures and oex are cut, deflect the A LR plates laterally and pin them flat to the silicone with a total of seven insect pins, two in the olfactory bulbs, two in the edges of the cut, cerebral protector commissure, two in the extended oex, and one in the midline of the spinal cord.Now using RNAs free reagents, fix the preparation in 4%para formaldehyde at room temperature for three hours with gentle agitation. The brains can then be analyzed by in situ two hybridization using the method described expression of neogen in transcripts was identified in spinal cord, projecting neurons of control, animals and in animals. Two weeks post lesion seen here is a representative uninjured control.After the complete spinal cord transection, there were changes in the expression of the neogen receptor. After two weeks, the neogen receptor in the non-control was preferentially expressed In bad regenerators like the M1 I one I two I four, or MTH neurons. The development of this technique paved the way for our ability to explore expression of guidance receptors, receptors for chondroitin sulfate, protio, glycans, and activation of cast bases all in the same brain.

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Learn complete spinal cord transection and brain dissection techniques in larval sea lamprey for studying axon guidance after injury. This method enables wholemount visualization of regenerating spinal-projecting neurons.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

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