Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (2023)


Here we describe the surgical procedures to perform a reliable lateral hemisection of the spinal cord (HX) in the ninthheAdult rat thoracic plane and neurobehavioral assessments to detect asymmetric deficits after such a unilateral lesion.


Incomplete spinal cord injury (SCI) often leads to sensorimotor impairment and is clinically the most common form of SCI. Brown-Séquard syndrome in humans is a common type of incomplete spinal cord injury caused by an injury to one half of the spinal cord, resulting in paralysis and loss of proprioception on the same (or ipsilesional) side as the injury. and loss of pain and temperature sensation on the opposite (or contralesional) side. Proper methods to create a lateral hemisection of the spinal cord (HX) and assess for neurological impairment are essential to establish a reliable animal model of Brown-Séquard syndrome. Although the lateral hemisection model plays a central role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateral function are lacking. The aim of this study is to describe step-by-step procedures to perform a rat lateral HX in the ninthhethoracic vertebral level (T9). We then describe a combined behavioral scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. Ranging from 0 to 18, the CBS-HX is made up of 4 individual scores including Unilateral Hind Leg Stepping (UHS), Docking, Contact Placement, and Grid Walking. In CBS-HX, the ipsilateral and contralateral hindlimbs are evaluated separately. We found that after a T9 HX, the ipsilateral hind paw showed impaired behavioral function, whereas the contralateral hind paw showed significant recovery. The CBS-HX effectively discriminated behavioral characteristics between ipsilateral and contralateral hindpaws and detected the temporal progression of ipsilateral hindpaw recovery. The CBS-HX components can be analyzed separately or in combination with other measures if necessary. Although we only provide visual descriptions of surgical procedures and behavioral assessments of a thoracic HX, the principle can be applied to other incomplete SCIs and at other levels of injury.


Incomplete spinal cord injuries (SCI) often result in severe and persistent sensorimotor impairment and are clinically the most common type of SCI1. Brown-Séquard syndrome in humans is caused by an injury to one half of the spinal cord, resulting in paralysis and loss of proprioception on the same (or ipsilesional) side as the injury and loss of pain and temperature sensation on the same side as the injury. the opposite side (or contralesional side). ) side2,3,4. Animal models with lateral hemisection of the spine are commonly used to mimic human Brown-Séquard syndrome and have been reported in rats.5,6,7,8,9, Zarigueyas10and monkeys7,11,12,13from different laboratories at different levels of the spine. However, detailed visualized procedures for making a standard lateral hemisection have not been described. Providing step-by-step procedures for a lateral hemisection should simplify the model and make it easier to compare or replicate experimental results in basic and translational research.

Unilateral SCI creates disproportionate and asymmetric behavioral deficits that are difficult to measure with traditional assessments for symmetrical injuries. An adequate methodology to assess neurological impairment in unilateral SCI is an essential part of developing a unilateral SCI model. Despite the central role of unilateral spinal injury, standardized protocols for assessing sensorimotor deficits in animals with such injury are lacking. The Basso-Beattie-Bresnahan (BBB) ​​movement rating scale is the most widely used post-SCI measure of function for adult rats.14giving a semi-quantitative description of locomotion as a whole. However, it does not measure each hind leg independently.

In this study, we report step-by-step procedures for preparing a rodent spinal HX on day 9.hethoracic vertebral level (T9). We are also introducing a Combined Behavioral Hemisection Scale (CBS-HX) that includes unilateral assessments of hindfoot gait (UHS), docking, contact placement, and grid walking to assess neurological impairment and recovery after unilateral SCI. . We hope that this model will be a useful model to study the mechanisms of injury and therapeutic efficacy in unilateral SCIs.


All surgical and animal handling procedures were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and the Institute for the Care and Use of Animals guidelines of the University School of Medicine. from Indiana.

1. General considerations

(Video) Philip Horner, PhD at the 2012 Spinal Cord Workshop

  1. For this study, use adult female Sprague-Dawley (SD) rats (weighing 200 g, n=12). Acclimate animals to all test environments and collect baseline data for all behavioral tests one week prior to surgery.
  2. Perform behavior ratings by two observers blinded to the experimental groups.

2. Animal preparation

  1. Clean the operating table with 70% ethanol. Place a preheated heating pad on the operating table. Cover the surgical area with a sterile surgical drape. Place the sterile gauze, cotton swabs, and autoclaved surgical instruments on the surface of the surgical drape.
  2. Turn on a bead sterilizer for intraoperative sterilization of surgical instruments.
    NOTE: An example of the tools used in this experiment is shown in the figure.illustration 1.
  3. Anesthetize the rat with an intraperitoneal (i.p.) injection of ketamine (87.7 mg/kg) and xylazine (12.3 mg/kg). Ensure that the correct level of anesthesia is achieved when there is no response to the toe pinch stimulus. Apply animal ointment to the animal's eyes to prevent drying of the cornea during surgery.
  4. Remove hair above the thoracic vertebrae by shaving (Figura 2A). Remove shaved skin with a vacuum equipped with a HEPA filter.
  5. Clean the surgical area with three alternating scrubs based on iodine and ethanol.
  6. Cover the animal with a sterile drape with a window over the intended incision site (Figure 2B). Note; In the video, the drape has been omitted for demonstration purposes.

3. Hemisection of the spine

  1. touch the 13heRib that is the lowest rib of the rat and a floating rib not connected to the sternum. Follow the 13heRib dorsally to identify its connection to the T13 vertebra, then count to identify the T10 vertebra.
  2. Use a scalpel blade (#15,illustration 1) to make a 3 - 4 cm midline skin incision on the back that is above 8-11hespinous processes of the vertebrae.
  3. Dissect bluntly under an operating microscope and separate the paraspinal muscles laterally from the spinous processes toward the facets of T9 and T10 vertebrae on each side using the same scalpel blade.
    NOTE: This approach separates the tissue very well without causing bleeding.
  4. Stabilize the spine with a modified stabilization brace. Make a cut on each side of the lateral vertebrae. Slide the stainless steel arms under the exposed facets of the transverse process and tighten the screws to ensure stability.
  5. Use a retractor to retract the muscles from the surgical site (Figure 2B) and to expose the T8-11 vertebrae and spinous processes (Figure 2C).
    NOTE: There is a large gap between the spinous processes of T8 and T9, which are landmarks for identifying T9 (Figure 2C, dorsal view). In lateral view, the spinous process of the T9 vertebra points caudally, the T10 spinous process points dorsally, and the T11 spinous process points rostrally; So, the 3 spinous processes form a pyramid and the T10 spinous process forms the apex (Figura 2D, vista lateral).
  6. Perform a dorsal laminectomy on the T9 vertebra with a rongeur. Cut the spinous process T9 and remove a small portion of the lamina to the left of the midline (Figura 3A, dashed line) and the entire right side of the sheet as laterally as possible (Figura 3A, Dashed line). For the laminectomy, carefully insert the rongeur under the blade, cutting a small piece of bone at a time until the desired laminectomy area is completed (Figure 3BYFigure 3C).
  7. Under an operating microscope, identify the dorsal midline of the spinal cord (Figure 3C). Insert a needle (30 G) vertically through the midline into the spinal cord with the beveled side toward the right side (Figure 4A).
    NOTE: The needle must penetrate the entire spinal cord to reach the ventral wall of the spinal canal.
  8. Stop any bleeding with a small piece of sterile gel foam.
  9. Insert one tip of an iridectomy/microsurgical scissors through the midline of the needle guide and the other tip along the lateral surface of the right hemifilament, then use the scissors to make a complete incision in the hemifilament. right (Figure 4B).
    NOTE: Use sharp micro scissors for spinal cord incision to minimize compression injury to the spinal cord during incision.
  10. Use the lateral edge of the same needle as a knife to cut the lesion space to confirm complete right hemisection. Check the integrity of the right hemisection by examining the floor of the spinal canal under the operating microscope (Figure 4C, cross-sectional view;Figure 4D, vista lateral;Figure 4E, dorsal view).
  11. Place a small piece of gelatin sponge over the lesion (Figura 4F). Using a cement mix, build a narrow bridge over the sponge and spinous processes of T8 and T10 (Figure 4G, H).
    NOTE: The purpose of using a cement bridge is twofold: 1) it separates the scar developed at the injury site from the rest of the tissue and 2) it facilitates detachment of the spinal cord segment after animal sacrifice.
  12. Sew the muscle and skin layers separately with 4-0 silk thread.
  13. Inject sterile 0.9% saline subcutaneously to maintain hydration. Injection of an analgesic buprenorphine (0.05-2.0 mg/kg S) 8-12 h/day subcutaneously for 2 days. Squeeze the bladder 2-3 times a day for the first week and 1-2 times a day for the next few weeks until spontaneous urination returns.

4. Postoperative care of animals

  1. Return the animal to its single-house cage of origin. Place wet rodent chow or gel in the bottom of the cage to help the animal eat/hydrate. Place a heating pad under the cage during postoperative recovery. Make sure the heating pad only covers half of the bottom of the cage to prevent overheating.
  2. Inject sterile 0.9% saline subcutaneously to maintain hydration. Injection of an analgesic buprenorphine (0.05-2.0 mg/kg S) 8-12 h/day subcutaneously for 2 days. Squeeze the bladder 2-3 times a day for the first week and 1-2 times a day for the next few weeks until spontaneous urination returns.

5. Evaluate the Unilateral Hemisection Seam (UHS)

NOTE: The Unilateral Hemisection Step (UHS) test is a direct measure of the ability of SCI animals to use their ipsilesional hind feet in the open field. As mentioned in 1.1, the animals were acclimated to an open field environment (42 inches in diameter).15twice a day for 7 days. Two observers blinded to the groups of animals perform the test. The UHS score is recorded both at baseline (7 days before T9 HX) and at time points after injury. The steps for the evaluation are described below.

  1. Place the animal in an open field setting and examine the animal's locomotion for 4 min.
    NOTE: During the test, the animal can be encouraged to exercise actively.
  2. With the form provided intabla 1, assign a score of 1 for yes and 0 for no to each behavior category, then add the total score for a final UHS score of 0 to 8.
    NOTE: According totabla 10: no movement of the hind legs is observed; 1 – 4: isolated movements of 3 joints of the hind legs (hip, knee and ankle); 5: sweep without weight support; 6: placement without weight bearing; 7: touchdown with weight support; and 8: pedaling assisted by weight.
  3. Collect UHS scores at both baseline (7 days before T9 HX) and post-injury time points.
    NOTE: Results are evaluated at different times after the T9 HX.

6. clutch

  1. Analyze CPL (gait coupling) with a video recording of an animal walking in a narrow track device or in a simple open field.
  2. In the area of ​​the clutchtabla 1, assign a score of 0 for No, 1 for Irregular/Awkward, and 2 for Fair for each CPL category.
    NOTE: The Linkage Test (CPL) is designed to assess the coordination of reciprocating limb movements, including homologous CPL (forelimbs-front/hindlimbs-posterior,Figura 5A), diagonal CPL (left forelegs-right hindlimbs or right forelegs-left hindlimbs,Figure 5B) and ipsilateral CPL (fore and hind limbs on the same side,Figure 5C). After a T9 HX, hindlimb deficits become visible on the ipsilesional side, leading to disruption of the homologous CPL (Figure 5D), CPL diagonal (Figure 5E) y CPL homolateral (Figure 5F).

7. Contact placement

NOTE: The hind paw contact placement test is used to assess the motor integration of hind paw responses to proprioceptive stimuli.sixteen. Proprioception is considered intact when the animal comes up to the surface with the hind foot after the hind foot has been dragged below the surface.

  1. Hold the animal upright so that both hind legs are available for the positioning response.
  2. Brush the dorsal surface of one hind foot slightly forward toward the edge of a surface (eg, an animal workbench).
  3. Note the placement of the feet on the surface and assign a score for the contact placement of the hind feet. 0: no placement; 1: Placement.
    NOTE: The dorsal surface is stimulated and subsequently the foot is extended and places the foot on the surface if the reflex is intact. The evaluation form is also included.tabla 1.

8. Walking the grid

(Video) Spinal Cord Injury

NOTE: The grid walk test assesses spontaneous motor deficits and limb movements involved in precise gait, coordination, and precise paw placement.

  1. Place a rat on a raised plastic-coated wire rack (36 × 38 cm with 3 cm2openings) and let him walk 30 steps freely across the platform.
  2. Count the total number of steps and the number of missteps for each limb. Video recordings will be made to confirm the count.
    NOTE: Two blinded observers assess forelimb and hindlimb paw placement when animals walk.
  3. Assign walk scores from the grid to each hindlimb as follows: 0: missteps greater than 15; 1: transfers less than or equal to 15; 2: errors less than or equal to 10; and 3: transfers less than or equal to 5.
    NOTE: Scoring is based ontabla 1. Cutoff scores are used as a measure of the severity of motor deficits.

9. Tissue Perfusion and Processing

  1. After adequate anesthesia similar to step 2.3, perfuse the animals carefully according to the transcardiac perfusion protocol.17.
  2. Dissect and collect the spinal cord samples and fix them in 4% PFA overnight.
  3. The samples can then be transferred to a 30% sucrose solution.
  4. Cut the spinal cord into transverse sections and stain selected sections with an axon marker SMI-31 and an astrocytic marker glial fibrillary acidic protein (GFAP) according to standard procedures.18.

representative results

The surgical procedures described above allow for the creation of a consistent and reproducible lateral HX on T9. After perfusion and skin removal, the surgical site at T9 could be easily identified by residual suture (Figure 6A). Further dissection allows exposure of the cementitious bridge (Figure 6B) and jelly cake (Figure 6C) in layers. The spinal cord is then exposed to the open spinal canal and a right lateral hemisection is confirmed (Figure 4D). The extent of the lesion can be further confirmed by its association with exposed vertebral bodies and ribs (Figure 6D). Immunofluorescence staining of a cross section at the epicenter of the lesion shows complete loss of the right cerebral hemisphere and preservation of the left cerebral hemisphere contralateral to the lesion. Section stained with an axon marker SMI-31 and an astrocytic marker glial fibrillary acidic protein (GFAP) (Figure 6E).

From a neurobehavioral point of view, the CBS-HX system is capable of detecting asymmetric deficits over time after a T9 HX. After HX, the ipsilateral hind paw lost the ability to walk, while the contralateral hind paw retained the ability to walk. For each behavioral measure, we performed 3 trials and used the mean of the 3 trials for quantification and analysis. We use the preoperative measurement as a reference, which we consider to be the most accurate control compared to using other rats. The scores of the 4 individual measures UHS, CPL, Contact Placement and Grid Walking can be evaluated separately (Figure 7A-D) or they can be combined into a composite CBS-HX (Figure 7E). Two-way ANOVA showed significant differences in UHS (F=23.199, p<0.001), docking (F=8.376, p<0.01), contact placement (F=17.672, p<0.001), grid run (F=19.261, p<0.001), CBS-HX (F=20.897, p<0.001) between the ipsilateral and contralateral sides.Figura 7Ashows UHS results after a T9 HX. In the first 3 days post-injury, the rats lost the ability to kick and received a score of 0-2 for the ipsilesional hind paw. Stepping movements began to appear on the ipsilesional side 7 to 10 days after injury, with most of the steps being dorsal. At 28 days post T9 HX, rats were able to perform plantar strides with nearly normal coordination, with an assigned UHS score of 8. In comparison, the contralesional hind paw was less disrupted and the UHS score fell within the first 5 days. after T9 HX and returned to baseline levels on day 10 post-injury. For the total CPL test (including homolateral, homologous, and diagonal coupling), both coordination stability and adaptability were significantly reduced after T9 HX (Figura 7B). 1-5 days post-injury, HX animals showed no signs of CPL. Over time, CPLs arose from the ipsilateral hind legs, often clumsily, unsteadily, and with inappropriate variations in speed, force, and direction. Contact mediation (Figure 7C) and walk grid (Figure 7D) of the ipsilateral hindlimbs were also affected by T9 HX, particularly within the first 5 days after injury, and generally recovered when the animal began to plantar pace. The CBS-HX Composite System includes UHS, CPL, Contact Placement, and Grid Walking tests for a maximum possible score of 18 (Figure 7E). Ipsilateral hindlimb motor function showed a decrease in CBS-HX scores after lateral T9-HX, consistent with deficits seen in human Brown-Séquard syndrome. Motor function of ipsilateral hindpaws showed a decrease in CBS-HX scores from 1 day to 4 weeks after T9 lateral HX compared to contralateral hindpaws (Figure 7E).

Therefore, the CBS-HX composite system, which combines UHS, CPL, contact placement, and lattice walking, can be used to assess the behavioral function of rats after lateral thoracic spinal cord injury for a maximum possible score of 18.

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (1)
Figure 1. Surgical instruments used to create a T9 right-sided hemisection. Please click here to view a larger version of this figure.

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (2)
Figure 2. Ex surgicalPose.A)Shave back hair covering the surgical region.B)Using a retractor, retract the muscles from the surgical site.C)Expose the T8-11 vertebral laminae and define the individual spinous processes (arrows). Note that there is a large gap between the spinous processes of T8 and T9, which is a landmark for identifying T9.D)The schematic drawing shows the lateral view of the spinous processes. Spinous processes T9-11 form a pyramid, with spinous process T10 forming the apex. Again, a large gap between the spinous processes of T8 and T9 is clearly seen as a landmark to identify T9 where a laminectomy will be performed.Please click here to view a larger version of this figure.

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (3)
Figure 3. Laminectomy and exposure of the right hemisphere.A)The schematic drawing shows the cross section of the spinal cord within the T9 vertebra. The dashed line indicates the extent of the laminectomy on each side.B)The schematic drawing shows the removal of a small part of the lamina on the left side and the entire vertebral arch on the right side. An arrow indicates the dorsal midline of the spinal cord.C)Dorsal view of the exposed spinal cord. Note that the dorsal vein was in the middle of the spinal cord, dividing the left and right hemichordia. The right hemisphere of the brain was completely exposed.Please click here to view a larger version of this figure.

(Video) Y3B Revision Lecture: Neurology

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (4)
Figure 4. Lateral hemisection.COMMERCIAL)Schematic drawings show midline needle insertion into the spinal cord(A),hemisection T9(B),the gelatin-cement sponge lining (C) and the side view of a T9 lateral hemisection(D).Dashed lines in C delineate the distant T9 vortex layer and the right hemicord.MI)Dorsal view of a hemisection of the right spinal cord.F)Place a small piece of gelatin sponge over the hemisection site.GH)A simple P cement bridge over the sponge and spinous processes of T8 and T10.Please click here to view a larger version of this figure.

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (5)
Figure 5. Coupling scheme (CPL)proof.The CPL test is designed to assess the coordination of alternate movements of the extremities, includingA)Homologous CPL (front-front/rear-rear members),B)Diagonal CPL (front left-rear right/front right-rear left) andC)Ipsilateral CPL (fore and hind limbs on the same side). After T9 HX (red inset, D–F), the hindlimb deficit became visible on the ipsilesional side and the animals showed incoordination on the homologue.(D),Diagonal(MI),y homolateral(F)CPL.Please click here to view a larger version of this figure.

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (6)
Figure 6. Tissue dissection and histology.After perfusion, the tissues were excised to expose the spinal cord. Cross sections were processed for double immunofluorescence staining for glial fibrillary acidic protein (GFAP, a marker for astrocytes) and SMI31 (a marker for axons).A)Suture exposure as a reference point for the lesion site (yellow arrow).B)Exposure of dental cementum (yellow arrow).C)Gelatin sponge exposure (yellow arrow).D)Identify the spinal hemisection on the right (yellow arrow).MI)A cross section of the spinal cord at the epicenter of the lesion, immunostained with GFAP (green) and SMI 31 (red). It shows that the right half of the spine was completely severed and the left half was well preserved.Please click here to view a larger version of this figure.

Lateral hemisection of the spinal cord and asymmetric behavioral assessments in adult rats | protocol (7)
Figure 7. Results of the neurobehavioral score.The diagrams show the results of the 5 measurements:A, the unilateral hemisection (UHS) value;B, embrague (CPL);C, mediation of contacts;D, walk in grid andmi, Combined Behavior Score (CBS) in the ipsilateral and contralateral hindpaws after a T9 HX. Data represent mean ± sem. *: p<0.05, **: p<0.01, ***: p<0.001 between ipsilateral and contralateral hindlimbs (two-way ANOVA, Tukey's multiple comparison test, n=12 rats/groups) .Please click here to view a larger version of this figure.

Subscore name/rangeDescriptionscore
Unilateral appearance of the hind legs.Slight observable movement of hind legs.NO0
(0-8)ankle movementNO0
knee movementNO0
hip movementNO0
sweep (without weight bearing)NO0
Placement (without weight support)NO0
Placement (with weight support)NO0
mediation of contactsNO0
wandering gridlose steps>150
Total CBS-HX

Tabla 1: Combined Behavioral Scores for Hemisection (CBS-HX)


In this study, we report stepwise procedures to produce a simple, consistent, and reproducible T9 spinal HX in adult rats that mimics Brown-Séquard syndrome in humans. We are also introducing a Combined Behavior Scoring System for Hemisection (CBS-HX) that can assess asymmetric neurological impairment and recovery progression, as measured by a combination of Unilateral Hind Leg Steps (UHS), Docking (CPL) , placement contact and grid. . Although we represent injury at the T9 level, this procedure can be easily and simply applied to other regions of the spinal cord, including the cervical and lumbar cords. We hope that this model, in conjunction with unilateral behavioral assessments, will be useful in studying the mechanisms of injury and therapeutic efficacy for this type of SCI.

Since the lateral HX model only injures the ipsilateral half of the spinal cord, the contralateral side of the spinal cord is largely spared and can be used as an internal control. Many ascending and descending tracts project unilaterally, and lateral hemisection in many circumstances results in damage to an axonal tract on one side and preserves the same tract on the opposite side, allowing comparison of the reorganization and functional consequences of these. tracts in the same animal. . Additionally, creating a more localized lesion may allow targeting to specific pathways. For example, a ventral and ventrolateral lesion can affect the reticulospinal and vestibulospinal pathways. A dorsal or dorsolateral lesion can affect the corticospinal and rubrospinal tracts. The hemisection or partial lesion model can also be used to study the anatomy and function of other pathways, such as the B. propriospinal, noradrenergic, or serotonergic pathways. Therefore, the hemisection model can be used in a unique way to study compensation by sensory afferents, by descending pathways, and by intrinsic spinal circuits. This model is also suitable for studying locomotor recovery mechanisms after HX.

Lateral HX leads to apparent behavioral disturbances that are evaluable under the motor task paradigm (eg, treadscan or treadmill) for automated gait analysis19. In addition, the conductance of the axonal pathways on the contralateral side of the lesion could be measured by electrophysiological recordings, and this evaluation offers the possibility of detecting functional reorganization after different treatments. In addition, unilateral injections of anatomical markers into neurons of a specific pathway allow visualization of anterograde labeled fibers crossing the midline and their connection with retrograde labeled neurons.20,21,22,23,24,25.

(Video) Management Guidelines in Neurotrauma by Dr Ved Prakesh, Neurosurgery

Although a typical spinal HX surgery takes less than 20 minutes, it takes practice to achieve a precise and consistent HX. First, it is important that spinal HX levels are consistent in all animals. Therefore, it is crucial that the appropriate vertebral segment for laminectomy is identified. Second, make sure the HX is complete. To make a full HX, a 30-gauge needle inserted vertically through the midline can be used to guide the incision with microscissors. Needle insertion also avoids damaging the posterior spinal vessels or cord through lesions. The second function of the 30 gauge needle is that it can act as a knife to trace the incision and ensure that the lesion is unambiguous. Third, the application of gelatin to the injury site can minimize the leakage of cerebrospinal fluid, and the application of the cement to the gelatin and the vertebral bed bridge can strengthen the stability of the spinal vertebrae at the injury site. injury and facilitate wound healing. To avoid signal interference when using electrophysiological recordings, the muscles, fascia, and skin should be sutured in layers with 4-0 silk thread. Finally, everything possible should be done to minimize damage to the contralateral spinal cord. Histologic review should be performed to confirm complete lateral hemisection on one side and preservation of the other half of the spinal cord on the other side (as shown inFigure 6E).

To improve locomotion after SCI, previous studies have used a wide range of strategies including cell transplantation and axon regeneration.8,18,26,27and activity-based rehabilitation28,29,30. Several behavioral tests have now been established to assess function and detect the best treatments after SCI. The BBB Movement Rating Scale was developed to assess musculoskeletal movement in symmetrical spinal injuries such as:14,31. Certain parameters of the BBB, such as B. Finger coordination and freedom are recorded by observing both hind feet. If a hind paw is intact and has other deficiencies such as those seen in asymmetric lesions, the intact hind paw will bias the evaluation of the affected hind paw. Since the BBB score after unilateral injury does not take into account the hind paw score of the other, it is not ideal for assessing unilateral spinal cord injuries. However, if joint movement and weight bearing are assessed separately on each side and not calculated as part of the BBB, the intact hind paw (similar to a sham control) will not confound the assessment of the affected hind paw. Also, the intact side will not affect the animal's overall score, as the intact hind paw does not show drastic deficits in joint movement, weight bearing, or stride.

The combined behavioral score for hemisection is intended to be a sensitive and easy-to-perform assessment of behavior recovery in the lateral hemisection rat model. It can be used to assess behavior in the early and late stages of recovery. The early phase is within 7-10 days after the injury. During the first 3 to 5 days post-HX, ipsilateral hindlimb activity increased steadily and should be assessed more frequently to record spontaneous or treatment-related recoveries in hindlimb movement. 5-7 days after HX, the rats began to perform sweeping movements of the hind legs without weight support. After 7 to 10 days, the rats began to stand up and walk normally. At this stage, attention should be paid to the pattern of steps. In the late phase (14 to 28 days), the activity of the ipsilateral hindlimbs was stable and almost normal.

Special attention should also be paid to the coupling capacity (CPL). The CPL (Gear Engagement) test can be performed with a video (eg tread/walk scan) or by filming a video during an open field test. The second option provides flexibility when investigators do not have access to the gait analysis system. A minimum of two consecutive touchdowns for each foot is required for both video recording sessions for this test. There are three coupling parameters for the analysis: homologous, homolateral and diagonal coupling (step 6.2). Each pairing includes a reference foot and the given foot. Take, for example, the homologous coupling (front left-front right or rear left-rear right), it is the first touchdown time of the given foot divided by a complete step time of the reference foot. Since the left and right feet must be out of phase, the perfect match must be 0.5. This is the same case with homolateral coupling (front left-rear left or front right-rear right). However, for diagonal coupling (left front-right rear or right front-left rear) the perfect coupling must be 0 or 1 since the two feet must be in phase. In step 6.4 we assign a score from 0 to 2 to each CPL. In detail, a score of 0 is intended to represent that the given foot cannot move to complete a touchdown, therefore there is no CPL; a score of 1 represents an erratic or awkward CPL because the given foot completes a touchdown but not in perfect engagement; a rating of 2 means a perfect fit of 0.5. The three concepts of coupling parameters are well described in the previous posts.32,33. The CPL can be combined with the Contact Placement and Grid Walk tests. Individual components of the combined behavior rating system will be more or less effective in different rat models of SCI. For CPL, the deficits were evident in the rate of change and the complete sequence. Hind paw placement proprioceptive deficits could be clearly revealed after unilateral HX. In our study, all rats showed deficits in ipsilesional hindpaw placement, whereas contralateral hindpaw placement did not show deficits. The grid walk test should be considered when contact placement involving the corticospinal tract begins to recover. To eliminate potential fatigue issues, the sequence of behavioral tests could be randomized for each test.

Finally, we report step-by-step procedures for creating a reproducible one.liveRat model of spinal T9-HX mimicking Brown-Séquard syndrome in humans. The combined behavioral assessment system for hemisection provides a more discriminatory measure of individual hindlimb behavioral outcomes to assess injury mechanisms and treatments following unilateral SCI. Although we provide only a visual description of the surgical procedures and behavioral assessments of a thoracic HX, the methods described here can be applied to other incomplete SCIs with varying degrees of injury.


We have nothing to disclose.


We thank Mr. Jeffrey Recchia-Rife for his excellent technical assistance. This work was supported in part by the Chinese PLA Jinan Military Region General Hospital Director's Foundation 2016ZD03 and 2014ZX01 (XJL and TBZ). Research in the Xu lab is recognized by NIH 1R01 100531, 1R01 NS103481, and U.S. Merit Review Award I01 BX002356, I01 BX003705, I01 RX002687. Grants from the Department of Veterans Affairs.


NameLook forCatalog numberComments
Hämostat Baby-MixerFST13013-14can be any brand
Raised Plastic Coated Wire Mesh GratingAny36×38 cm by 3 cm2openings
gel foamdoctor moore2928It can be any brand.
Kit grip cement, powder and solventDentsply675570It can be any brand.
Microperlene sterilizerFSTHEcan be any brand
pearson's rodentFST16015-17It can be any brand.
RetractorJinxie Surgical Tools6810can be any brand
scalpel handleFST10003-12can be any brand
Simple-P-ZementString instrumentsIt can be any brand.
TreadScan automatic gait analysisCleverSys Inc.HEcan be any brand



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