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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</ol>

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.

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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

Research

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

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Behavior

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Medicine

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Bioengineering

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

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

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

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

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...