This work was supported by the International Spinal Research Trust and the Rosetrees Trust.

Ethics statement: All procedures were in accordance with guidelines from the UK Home Office and Animals (Scientific Procedures) Act of 1986.
1. Surgery and Injury
NOTE: Using rats heavier than 220 g can help with postoperative recovery if the surgeon finds animals often lose more than 10% of their preoperative body weight.
<ol>
	<li>Before surgery, test the animals for their forepaw preference with the cylinder rearing test 15 or the Montoya staicase test 24. Preferably, cut the pyramid/ the CST corresponding to the dominant forepaw. 
	NOTE: For two days prior to surgery, place the following items into the cages, so that the rats become familiar with food items that will be beneficial during recovery from surgery: hydrogel packs, wet mash, and high caloric food supplements. Sterile versions of these food supplements are available from various vendors.</li>
	<li>Anesthetize female Lister hooded rats (weighing between 200 and 250 g) with isoflurane (5% for induction and 1 to 2% for maintenance) in oxygen (flow-rate: 1.5 L/min). &#8232;NOTE: The choice of anesthetic is important. Inhalation anesthetic allows for faster regulation of the depth of anesthesia than ketamine, which is also commonly used. Isoflurane allows for better control of the breathing frequency and depth. The choice of anesthetic is isoflurane. NOTE: Using rats heavier than 220 g can help with postoperative recovery if the surgeon finds animals often lose more than 10% of their preoperative body weight.</li>
	<li>Administer Carprofen subcutaneously as an analgesic when the animals are anaesthetized to reduce inflammation and to provide analgesia. Note: Carprofen is given during pyramidotomy surgery in our laboratory according to our veterinarian&#39;s advice. Opioids might cause further respiratory depression during surgery. </li>
	<li>Maintain the animals at 37 &#176;C during surgery using a homeothermic blanket system and a rectal temperature probe.</li>
	<li>Confirm full anesthesia by checking for the paw pinch withdrawal reflex and the blink reflex. 
	Note: Optional: To counter respiratory problems during surgery, intubation and ventilation might be feasible. However, bear in mind that the trachea needs to be displaced during surgery.</li>
	<li>Once anesthetized, place the rat in a supine position, shave the ventral neck and sterilize it with 1% chlorhexidine wipes and/or alcohol wipes. Apply a sterile surgical blanket over the animal and keep the area sterile at all times.</li>
	<li>Once anesthetized, place the rat in a supine position, shave the ventral neck and sterilize it with 1% chlorhexidine and alcohol wipes. Apply a sterile surgical blanket over the animal and keep the area sterile at all times. 
	NOTE: Optionally, the rat&#8217;s head can be fixed in a stereotaxic frame in the supine position during surgery to disable mobility of the head, i.e. the occipital bone.</li>
	<li>Make a 2 to 3 cm long midline incision from the chin to almost the rostral end of the sternum using a sterile scalpel (#10). Optionally, apply small bulldog clamps to retract the skin.</li>
	<li>Blunt dissect the upper layers of tissue such as glands (e.g. submaxillary gland and parotid gland) and muscles covering the trachea using reverse action with blunted scissors and toothed forceps. Always stay on the midline. The tissues should separate easily.</li>
	<li>Once the trachea is exposed, displace it to one side. The midline is visible underneath it with two white fat pads, marking the midline at the rostral end.</li>
	<li>Rostral to the white fat pads, blunt dissect tissue until the ventral surface of the skull (the basioccipital bone) is reached.</li>
	<li>Insert long-toothed retractors (tooth length 5.5 mm or 16 mm) to keep the trachea displaced to one side (maximum 1 cm) and to expose the base of the skull. Displacement of the trachea can obstruct breathing. 
	NOTE: For some animals, loosen the retractors at multiple times during the surgery to remove the strain on the trachea, e.g. every 10 min for 2 min. Hook retractors can be used instead of long-toothed retractors to reduce the strain of displacement.</li>
	<li>Carefully cauterize small visible blood vessels at the rostral end going towards the larynx to avoid bleeding. 
	NOTE: In order to prevent cauterizing the recurrent laryngeal nerve blood vessels are carefully pulled away from the tissue wall before they are cauterized.</li>
	<li>Adjust the microscope for the remainder of the surgery.</li>
	<li>Remove the periosteum covering the base of the skull with fine forceps. Adjust the retractors to achieve the best possible view for drilling.</li>
	<li>Feel the uneven surface of the basioccipital bone with fine forceps, noting an elevation at the midline, which covers the basilar artery. Rostrally, the skull becomes raised as a slight convexity.</li>
	<li>Depending on which side the lesion is desired, drill a hole roughly 1 mm lateral to the midline in mediolateral movements. 
	NOTE: Remember that the rat is in the supine position, and its left side may be on the investigators right hand side!</li>
	<li>Once a hole is made, enlarge towards the midline with small vertical up-and-downwards-movements with the drill until the basilar artery is clearly visible and enlarge laterally for at least 2 mm.</li>
	<li>Make sure the basilar artery is clearly visible. The pyramid can be identified by its slightly bulging shape (Figure 2) and by bordering blood vessels.</li>
	<li>Remove any remaining bone fragments with fine forceps.</li>
	<li>Apply one drop of Doxapram hydrochloride, a respiratory stimulant, on the tongue.</li>
	<li>Open the dura longitudinally with a 26 gauge needle and fine forceps lateral to the basilar artery. Soak up the cerebrospinal fluid (CSF) and any bleeding with cotton buds.</li>
	<li>Avoiding injury to the basilar artery, make a cut approximately 1.5 mm wide spanning the width of the pyramid and 0.5 mm deep with Vannas Spring Scissors perpendicular to the basilar artery (Figure 2) to interrupt the CST fibers. 
	NOTE: Mark the microscissors beforehand at 0.5 mm from the tips. Once the dura is opened the basilar artery can be moved slightly to one side allowing better access to the pyramid.</li>
	<li>Repeat the cut using the tip of a 26 gauge needle to ensure the lesion includes the fibers close to the basilar artery.</li>
	<li>Stop the bleeding with light pressure applied by cotton buds. Optionally, the cut can be covered with gelfoam.</li>
	<li>Remove the retractors, replace the tissues and suture only the skin with 3-0 Vicryl sutures.</li>
	<li>After the surgery, keep the animal in an incubator at 32 &#176;C until fully awake and administer 5 ml saline subcutaneously, if required. Do not leave the animal unattended.</li>
	<li>Give Carporfen subcutaneously as an antiinflammatory and as an analgesic for the post-surgical pain one day postsurgery.</li>
	<li>Monitor animals closely for one week after surgery and at least once a week thereafter.</li>
	<li>As required after surgery, place hydration gel packs, pureed baby food, wet mash and dry chow on cage floor. Provide water bottles with long spouts (lacking ball bearing valves). </li>
</ol>

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The authors have nothing to disclose.

We have described the pyramidotomy injury model. The corticospinal tract is cut at the level of the brainstem. From a technical point of view this surgery requires a lot of cautiousness and accuracy. The brainstem accommodates the breathing centers and the cardiovascular centers in multiple nuclei such as the nucleus ambiguous and solitarii embedded in the reticular formation underneath the pyramids. If the cut is made too deep this can lead to sudden breathing difficulties, which may be fatal. We give Dopram just before the cut to increase the breathing activity to prevent a sudden breathing cessation. If the breathing stops it is worth ventilating the animal for the following five minutes with oxygen as it may recover. Additionally, any damage to the basilar artery should absolutely be avoided. Complications during the first few days postinjury are: another hemorrhage in the brainstem affecting vital functions, considerable weight loss, severe breathing difficulties due to trachea displacement and severe orientation and balance problems of the animal due to extensive injury including the olives just lateral to the pyramids. These complications can be treated as end-points and animals may be humanely killed. Close monitoring during the first week after surgery is essential. Supplementary food can be given in order to counterbalance weight loss. Airway inflammation (raspy breathing) normally resolves within one to two days. Generally, younger animals are easier to operate on as their neck is less deep. Furthermore, they are more resilient and have less breathing complications.
It requires some practice to achieve a complete lesion of the CST with a pyramidotomy. Similarly, in two studies by Steward et al. 16, which set out to replicate a former experiment by Benowitz et al. 28, reported initially high mortality rates and that some of their lesions were incomplete. Behavioural deficits will also be variable. We recommend that a new surgeon performs many practice pyramidotomy surgeries before any full experiment to test a candidate therapy is performed. Ways to prevent incomplete lesions are retracing the cut with a hypodermic needle making sure the corticospinal fibers close to the basilar artery are severed as described in this protocol. Other options are to aspirate the lesion site with a tipped glass suction pipette as described in Zhou et al. 2003 29. A fine tungsten wire can also be used to cut the tract 22. An alternative to pyramidotomy is to cut the cervical C3 dorsal CST unilaterally and then to cut with a ventral approach the ipsilesional C2 ventral part of the CST 18. However arguably, a complete lesion is not necessary if the goal is to study sprouting of the contralesional CST; if the majority of the pyramid is lesioned and a sprouting response from the unlesioned side occurs this can be sufficient.
One aim of the pyramidotomy lesion model is to ablate one side of the corticospinal tract and use neuroplasticity-inducing therapies to stimulate the plasticity and sprouting of the intact non injured fibers. To test neuro-protective therapies this model is unsuitable and other injury models such as contusion injuries should be used. Outcomes of pyramidotomies are assessed most often with behavioural testing and anatomy. The horizontal ladder test, the cylinder rearing test and Montoya staircase test are the most commonly used behavioural assessments. Other tests described in the literature are the pellet reaching test, rope climbing test, gait analysis and the sticky tape test 3,17-19. However, the outcomes of most behavioural test are often only monitored up to a maximum of 42 days postlesion 3,4,11,17,18,20-22. From past experiences, rodents recover most function at later time points without any treatment intervention. We have shown that the horizontal ladder test and the Montoya staircase pellet reaching test detect disabilities up to a later time point. They can be used to measure functional recovery due to a treatment. The lesion completeness is judged by PKC gamma staining (which labels CST fibers in the dorsal columns) of the spinal cord at rostral cervical level or eriochrome cyanine staining of transverse sections of the lesion site. Biotinylated dextran amine can be injected into the contralesional cortex to assess the sprouting of the intact fibers across the midline into the denervated spinal cord area.
In conclusion, the pyramidotomy model is good for assessing neuroplasticity-inducing abilities of a treatment drug for central nervous system injuries if the surgery is successfully mastered. It assesses plasticity of intact fibers. The next step would be to use a more clinically relevant model such as contusion injuries to assess the drug&#8217;s ability to overcome larger injuries affecting multiple tracts.

The corticospinal tract (CST) is one major motor tract in the human spinal cord. Damage to this tract after spinal cord injury results in considerable loss of dexterity. The CST is especially important for fine motor movement in humans, such as digit movement and distal muscle control. It is also found in rodents although its anatomical location within the spinal cord is different to that of humans and CST damage is less disabling 1-4.
One in vivo model of CST injury involves unilateral pyramidotomy, where one pyramid in the ventral brainstem is cut rostral to the decussation (Figure 1). The pyramids are a clear anatomical feature on the ventral side of the medulla oblongata through which the CST runs before it decussates. In rats, the majority of fibers (around 95%) decussate at the caudal end of the medulla and are thereafter found in the dorsal medial part of the spinal cord. However, approximately 10% of the decussated fibers run in the dorsolateral columns. The remaining ~5% of fibers stay uncrossed on the ipsilateral side and travel in the ventral medial part 5,6. After pyramidotomy the spinal cord loses its direct input from the contralateral CST (Figure 1).
The pyramidotomy model is particularly good for testing the responses of the intact fibers of the unlesioned CST to a treatment. It allows assessment of plasticity and sprouting of the intact fibers across the midline in the spinal cord, which innervate the denervated areas. The corticospinal tract has been observed to sprout considerably in spinal injury models and mediates especially forelimb recovery 7-10. A treatment might aim to enhance this forelimb recovery. The efficacy of a therapy can be tested with behavioural assessments, anatomical tracing techniques of the unlesioned tract and terminal electrophysiological experiments 3,11,12.
The advantage in comparison to most other preclinical models of spinal injuries is that it specifically affects one tract compared to other spinal injuries often affecting multiple tracts. Only one tract in a clear, superficial anatomical location is lesioned. Another advantage is that the pyramidotomy is a reproducible injury model. It is a suitable in vivo resource for researchers to test the effect of potential neuroplasticity-inducing therapies, which aim at recovery of motor function after injuries or disorders affecting the motor system 13,14.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Carprofen</td>
			<td>Norbrook</td>
			<td>Vm No; 02000/4229</td>
			<td>give 5mg/kg twice daily</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Harvard Instruments</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors- Tough Cut</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11019-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Long-toothed Alm retractors</td>
			<td>Fine Science Tools</td>
			<td>17009-07</td>
			<td></td>
		</tr>
		<tr>
			<td>cautery system kit</td>
			<td>Harvard Instruments</td>
			<td>726067</td>
			<td></td>
		</tr>
		<tr>
			<td>fine Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
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			<td>Carbon Steel burrs</td>
			<td>Fine Science Tools</td>
			<td>19007-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopram V-drops</td>
			<td>Pfizer</td>
			<td></td>
			<td>apply 1 drop to tongue</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
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		<tr>
			<td>gelfoam</td>
			<td>Equimedical</td>
			<td>EQU705001</td>
			<td></td>
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			<td>3-0 Vicryl sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>operating microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Anaesthesia System Isoflurane K/F Single Gas</td>
			<td>VetTech Solutions</td>
			<td></td>
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		</tr>
		<tr>
			<td>Scales</td>
			<td>Ohaus</td>
			<td>NVT3201</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel Scalpel blades No. 10</td>
			<td>Swann-Morton</td>
			<td align="right">201</td>
			<td></td>
		</tr>
		<tr>
			<td>25g needles</td>
			<td>Terumo</td>
			<td>NN-2525R</td>
			<td></td>
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			<td>syringes (1ml and 5ml)</td>
			<td>Terumo</td>
			<td>SS+01T1 / SS*05SE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (Sodium Chloride 0.9%)</td>
			<td>Fresenius Kabi</td>
			<td>Pl 08828/0178</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton buds</td>
			<td>Johnson and Johnson</td>
			<td align="right">5000207582502</td>
			<td>sterilize before use</td>
		</tr>
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</table>

unilateral pyramidotomy of the corticospinal tract in rats for assessment of neuroplasticity-inducing therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

Survival rates.
In a representative study conducted by us we had a survival rate of 16 out of 20 female Lister hooded rats (200 to 250 g). The most common complication is breathing difficulties because of the trachea displacement or damage to the respiratory centers in the brainstem just dorsal to the pyramids (nucleus solitarii, ambiguous and parabrachilis). Similar results have been reported in the literature 16.
Behavioural outcomes.
A range of behavioural tests have been used to assess sensorimotor outcome after pyramidotomy in rodents: the horizontal ladder test, the single pellet reaching test, the cylinder rearing test and Montoya staircase test are the most commonly used behavioural assessments. Other tests described in the literature are the pellet reaching test, rope climbing test, gait analysis and the sticky tape test 3,17-19. Most of these show a functional motor deficit 1 week after injury. Currently, most studies described in the literature only perform behavioural testing up to 42 days 4,11,13,20,21, or for a shorter time 3,18,22. We have performed behavioural testing for a prolonged period of time and have seen essentially full recovery of motor function 8 weeks post pyramidotomy for some of the behavioural tests, except the Montoya staircase test and the horizontal ladder test. This is probably for several reasons. Firstly, the injured CST rostral to the lesion may form synapses on neurons in the brainstem that act as indirect relays to motor neurons in the spinal cord. Secondly, the uninjured (contralateral) CST may sprout and forms synapses on neurons in the brainstem or spinal cord that act as relays to motor neurons. Thirdly, other spared motor tracts such as the reticulospinal, vestibulospinal or rubrospinal tracts may take over some function. Thus, a lasting treatment effect is difficult to detect with many behavioural tests in the pyramidotomy model. Some behavioural testing in this model however could indicate accelerated recovery early after the injury, which is related to a treatment.
Cylinder Rearing test 3,4,23
Based on the cylinder rearing test the CST corresponding to the dominant forepaw was injured. Rats are placed in a plexiglass cylinder and rearing behaviour is observed for a period of 3 min. Preinjury there was a slight preference for the use of one forepaw, the dominant forepaw, during vertical exploration (Figure 3A). The injury affects the dominant side and the use of the contralesional forepaw decreased to 28% at 1 week postinjury (Repeated Measures ANOVA p &#60;0.05; post-hoc analysis revealed animals have significant deficits at Week 1, 2 and 3, p &#60;0.05, Fisher&#8217;s LSD). 4 weeks postinjury the animals recovered and used the affected forelimb for 45% of rears, and after 6 weeks this was 57% (n = 16, mean &#177; standard error is shown). Similarly, Starkey et al. (4) have assessed mice using the cylinder test for up to 42 days after pyramidotomy and reported a significant decrease in the use of the affected forepaw throughout the whole testing period.
Montoya Staircase test 24
Rats require pretraining for this test to acclimatize prior to injury. To increase motivation during the testing session animals are food restricted to 15 g food per rat the night before. The food deprived rats are placed in the Montoya staircase for 15 min. On each side, there is a staircase of 7 steps with wells, each containing 3 pellets. The total number of retrieved pellets on each side are recorded. The number of retrieved pellets with the contralesional/affected forepaw decreased significantly from 61% preinjury to 3% 3 days postinjury (Figure 3B). The ability to retrieve pellets was still significantly impaired at 4 weeks postinjury with 20% of pellets retrieved. Rats show a significant deficit up to week 8 with only 29% of the pellets eaten at that time point (Repeated Measures ANOVA p &#60;0.05, post hoc analysis revealed animals have significant deficits on the contralesional side at all weeks, p &#60;0.05, Fisher&#8217;s LSD). The ipsilesional forepaw is affected up to 1 week postinjury; however this recovers by the second week (Repeated Measures ANOVA p &#60;0.05, post hoc analysis revealed animals have significant deficits at Day 3 and Week 1 on the ipsilesional forepaw, p &#60;0.05, Fisher&#8217;s LSD). The initial deficits of the ipsilesional forepaw might be explained by the lesioning of the ipsilateral portion of corticospinal tract running through the cut pyramid, with subsequent sprouting and recovery.
Horizontal Ladder test 25
Rats require pretraining for this test. Rats walk three times over a 1 m long ladder with irregular rung spacing. Videos are analysed later in slow motion and the total number of foot slips and misses for each paw is quantified. The graph in Figure 3C represents the total number of misses and slips of the affected and less affected side (forepaw + hindpaw) as a percentage to the total number of steps taken. Rats have a significantly increased number of errors 1 week postinjury with 10% or 11% on the affected side and less affected side respectively. This deficit is persistent on the contralesional/affected side up to week 8 with 10% errors made, however, resolves on the ipsilesional/less affected side by 2 weeks (Repeated Measures ANOVA, post hoc analysis revealed animals have significant deficits at all weeks on the contralesional side and significant deficits at Week 1 and 3 on the ipsilesional side, p &#60;0.05, Fisher&#8217;s LSD).
Electrophysiological testing 
In a terminal electrophysiological set-up the animal was anaesthetized intraperitoneally with urethane. Throughout the procedure, it was kept at 37 &#176;C &#177; 1 &#176;C. Its ventral neck region, its forelimbs and chest region were shaved and disinfected with iodine scrub. The pyramids were exposed as previously described and the radial nerve was exposed in the upper arm by a ventral approach incising the skin and the pectoralis major. The distal end of the radial nerve was cut and the nerve was laid over two silver-wire hook-electrodes in a mineral oil bath. Either pyramid was stimulated with concentric bipolar electrodes at different depths with 5 pulses at 300 Hz and increasing stimulation amplitude. Recordings were always made from the radial nerve of the disabled arm (Figure 4).
Uninjured animals had a strong response at latencies between 12 to 20 msec in the radial nerve when the contralateral pyramid was stimulated (Figure 4B). Ipsilateral stimulation only results in occasional activation (Figure 4C). Animals, which have previously received pyramidotomies, on the contralateral side to the recording arm show no response in the radial nerve when the lesioned tract is stimulated above the lesion (Figure 4B&#8217;). However, stimulation of the CST fibers in the pyramids ipsilateral to the recording electrodes resulted in a stronger activation in the radial nerve compared to uninjured animals at 12 weeks postinjury (Figure 4C&#8217;).
Anatomical outcomes.
Animals were euthanized 10 weeks postsurgery according to Schedule 1 of the Animals (Scientific Procedures) Act 1986. Two weeks previously, the uninjured CST was traced with biotinylated dextran amine (BDA) injections into the motor cortex 26 ipsilateral to the disabled arm (Figure 1). This allows quantification of uninjured fibers, which cross the midline and supply the contralesional spinal cord (Figure 5C, D). Uninjured animals only have a few fibers crossing the midline, whereas this increases in response to injury. Transverse cervical spinal cord sections were stained with protein kinase C (PKC) gamma to assess the proportion of denervation in the lesioned CST (Figure 5E)16. Alternatively, the completeness of the lesion can be assessed by injecting BDA tracer into the motor cortex on the ipsilesional side of the pyramidotomy injury. The absence of labeling in the contralateral dorsomedial CST and the ipsilateral ventromedial CST provides evidence of complete unilateral transection.
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/51843/51843fig1highres.jpg" width="400" /> 
Figure 1: Schematic showing the corticospinal tracts in the rat and unilateral axotomy of the CST in the pyramids. Top image: Corticospinal neurons originate in the cortical layer 5 pyramidal cells. Biotinylated dextran amine (depicted in green) is injected into the contralesional cortex two weeks prior to the end of the experiment. Middle image: The tracts form the pyramids on the ventral surface of the medulla and at the spinomedullary junction the majority of axons decussate. Unilateral pyramidotomy is performed in the caudal medulla (shown in red). Lower image: The majority of the corticospinal tracts run down in the dorsomedial and dorsolateral parts of the spinal cord. A minority stays ipsilaterally and runs ventromedially.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig2highres.jpg" width="500" /> 
Figure 2: Ventral view of the rat brain. The pyramids are distinctive because they are raised, parallel convexities (yellow box). The borders of the pyramids are defined medially by the basilar artery (bas) over the midline and laterally at the base of the convexity halfway between the basilar artery and the paraolivary artery (pol) as seen in (C). Craniotomy is performed over the basilar artery and lateral extending to the paraolivary artery (red box in C). One pyramid is cut perpendicularly to the basilar artery (red line in C, cut is shown on specimen in A and B). Lesions placed too caudally near the vertebral arteries (vert) will be incomplete because the CST starts to decussate and because the vertebral artery is set off the midline. (C) Image adapted from &#8220;The Rat Nervous System&#8221; by George Paxinos27. NOTE: This Figure shows a lesion on the left hand side of the images: this is the animal&#8217;s right pyramidal tract because it is lying supine. Please note however that the video shows a lesion to the animal&#8217;s left pyramidal tract.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig3highres.jpg" width="500" /> 
Figure 3: Representative results for the cylinder rearing test, Montoya staircase pellet reaching test and Horizontal ladder test. There is a motor deficit detected by all tests after injury. However, no deficit can be detected with the cylinder rearing test at 4 weeks postinjury, whereas sustained deficits can be detected with the Montoya staircase test and the horizontal ladder test up to 8 weeks postinjury (n = 16 per group, means and standard error are shown). Asterisk indicates p &#60;0.05 relative to preinjury baseline (Fisher&#8217;s LSD test).<a href="https://www.jove.com/files/ftp_upload/51843/51843fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig4highres.jpg" width="500" /> 
Figure 4: Electrophysiology assessing the corticospinal tract before and after pyramidotomy. (A) shows the set-up for the terminal experiment. We stimulate the pyramids with a train of 5 pulses at various depths rostral to the site of lesioning and decussation. The red tract is the lesioned tract. We record from the radial nerve on the contralesional side. (B) and (C) show example recordings with ipsilesional stimulation or contralesional stimulation before or 12 weeks after pyramidotomy. Ipsilesional stimulations evoke multiple compound action potentials (spikes in the trace) in the radial nerve before injury (B). These are abolished by the injury and activity does not return within 12 weeks (B&#8217;). Contralesional stimulation evokes rare single compound action potentials preinjury (C). 12 weeks after injury activity considerably is increased (C&#8217;), possibly due to intact axons sprouting across the midline and plastic changes within the spinal cord. <a href="https://www.jove.com/files/ftp_upload/51843/51843fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig5highres.jpg" width="500" /> 
Figure 5: Anatomical evaluation after pyramidotomy. (A, B, C, D) Biotinylated dextran amine was used to trace the corticospinal tract by injecting it into the motor cortex corresponding to the unlesioned CST (see green in Figure 1). The dorsal medial, the ventral medial components of the CST are clearly visible (A, dorsal medial part magnified in B). (C) and (D) are magnifications from (A) of fibers sprouting across the midline into the grey matter of the denervated side in response to a treatment. To assess the completeness of the lesion transverse sections of the cervical level 4 (12 weeks postinjury tissue) were cut and stained with antibodies against PKC gamma (E). The CST on the left hand side of the image is intact and can be seen bulging across the midline into the denervated side, presumably because of the structural disintegration of the injured dorsal CST.

Watch this Scientific Journal Video about Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies at JoVE.com

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

unilateral-pyramidotomy-corticospinal-tract-rats-for-assessment

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JoVE Journal

Neuroscience

15.8K Views.  King's College London. The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (“pyramidotomy”) and postoperative assessments are described in this protocol.

Video: Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential for recovery based on clinical assessments alone. The functional integrity of the corticospinal tract is an important prognostic biomarker for recovery of UL function, particularly for those with severe initial UL impairment. This article presents a protocol for evaluating corticospinal tract function within 1 week of stroke. This protocol can be used to select and stratify patients in trials of interventions designed to improve UL motor recovery and outcomes after stroke. The protocol also forms part of the PREP2 algorithm, which predicts UL function for individual patients 3 months poststroke. The algorithm sequentially combines a UL strength assessment, age, transcranial magnetic stimulation, and stroke severity, within a few days of the stroke. The benefits of using PREP2 in clinical practice are described elsewhere. This article focuses on the use of a UL strength assessment and transcranial magnetic stimulation to evaluate corticospinal tract function.

This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential ...