A novel experimental model of cervical spondylotic myelopathy (CSM) to facilitate translational research

Neurobiology of Disease 54 (2013) 43–58

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

A novel experimental model of cervical spondylotic myelopathy (CSM) to facilitate translational research Spyridon K. Karadimas a, b, Eun Su Moon b, Wen-Ru Yu b, Kajana Satkunendrarajah b, Joannis K. Kallitsis c, Georgios Gatzounis d, Michael G. Fehlings a, b, e, f,⁎ a

Institute of Medical Sciences, Faculty of Medicine, University of Toronto, Ontario, Canada Division of Genetics & Development, Toronto Western Research Institute, and Spinal Program, Krembil Neuroscience Centre, University Health Network, Toronto, Ontario M5T 2S8, Canada Department of Chemistry, University of Patras, Patras 26500, Greece d Department of Neurosurgery, University of Patras, Patras 26500, Greece e Neuroscience Program, University of Toronto, Toronto, Ontario M5T 2S8, Canada f Department of Surgery, Division of Neurosurgery, University of Toronto, Toronto, Ontario M5T 2S8, Canada b c

a r t i c l e

i n f o

Article history: Received 16 August 2012 Revised 1 February 2013 Accepted 19 February 2013 Available online 4 March 2013 Keywords: Cervical spondylotic myelopathy CSM Chronic cervical spinal cord compression Nontraumatic cervical spinal cord injury

a b s t r a c t Cervical spondylotic myelopathy (CSM) is the most common form of spinal cord impairment in adults. However critical gaps in our knowledge of the pathobiology of this disease have limited therapeutic advances. To facilitate progress in the field of regenerative medicine for CSM, we have developed a unique, clinically relevant model of CSM in rats. To model CSM, a piece of synthetic aromatic polyether, to promote local calcification, was implanted microsurgically under the C6 lamina in rats. We included a sham group in which the material was removed 30 s after the implantation. MRI confirmed postero-anterior cervical spinal cord compression at the C6 level. Rats modeling CSM demonstrated insidious development of a broad-based, ataxic, spastic gait, forelimb weakness and sensory changes. No neurological deficits were noted in the sham group during the course of the study. Spasticity of the lower extremities was confirmed by a significantly greater H/M ratio in CSM rats in H reflex recordings compared to sham. Rats in the compression group experienced significant gray and white matter loss, astrogliosis, anterior horn cell loss and degeneration of the corticospinal tract. Moreover, chronic progressive posterior compression of the cervical spinal cord resulted in compromise of the spinal cord microvasculature, blood–spinal cord barrier disruption, inflammation and activation of apoptotic signaling pathways in neurons and oligodendrocytes. Finally, CSM rats were successfully subjected to decompressive surgery as confirmed by MRI. In summary, this novel rat CSM model reproduces the chronic and progressive nature of human CSM, produces neurological deficits and neuropathological features accurately mimicking the human condition, is MRI compatible and importantly, allows for surgical decompression. © 2013 Elsevier Inc. All rights reserved.

Introduction Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord dysfunction among adults over the age of 55 (Young, 2000) and the key underlying risk factor predisposing patients to traumatic central cord syndrome, the most common cause of cervical spinal cord injury (SCI) (van Middendorp et al., 2010). Indeed, 25% of spinal cord dysfunction in the U.K. is caused by CSM (Moore and Blumhardt, 1997). Critical gaps in our knowledge of the pathobiology of CSM have limited therapeutic advances for this common cause of ⁎ Corresponding author at: Gerald and Tootsie Halbert Chair in Neural Repair and Regeneration, Toronto Western Hospital, University Health Network, West Wing, 4th Floor, Room 4W-449, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. Fax: + 1 416 603 5298. E-mail address: [email protected] (M.G. Fehlings). URL: http://www.drfehlings.ca/ (M.G. Fehlings). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2013.02.013

neurological dysfunction. While surgical intervention can attenuate the progression of CSM, most patients are still left with significant neurological impairment (Fehlings et al., 2012). Remarkably little translational research has been directed at developing pharmacological and biological approaches to improve outcomes for this condition. The lack of reliable rodent models of CSM has been a clear limitation to advancing the field. To date, various attempts have been made to reproduce CSM in animal models (Karadimas et al., 2010; Klironomos et al., 2011; Lee et al., 2012). Although some of these studies have significant merit, they also have critical limitations. Most of these studies are acute or subacute in design and consequently fail to model the chronic and progressive nature of the disease. Moreover, many of these models do not accurately reproduce the main human neuropathological and clinical features of CSM, are not MRI compatible and, do not facilitate surgical decompression. These limitations and the lack of neuroprotective treatments for CSM point toward the need for a novel model. This model

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should result in slow, progressive compression, should produce neurological deficits that mimic the human condition, should ideally involve the mid-cervical cord segments and should allow for surgical decompression. In this study we report, for the first time, a unique, clinically relevant model of CSM in rats. It is less invasive than previous models, reproduces the clinical symptoms and the neuropathological features of the human disease, and unlike previous models, is MRI compatible and allows for surgical decompression. Materials & methods Compression material A synthetic aromatic polyether was used as the compression material. Its most characteristic property is the capacity to absorb phosphate anions, increase calcium phosphate sedimentation and induce new osteoid formation (Klironomos et al., 2011). Experimental groups & surgical procedures A summary of the experimental groups is depicted in Table 1. All animal protocols were approved by the animal ethics committee of the University Health Network, Toronto, Ontario, Canada. Material implantation Female Sprague–Dawley rats (weight 300–400 g) were anesthetized with 2% isoflurane with oxygen and N2O. The surgical area was shaved and disinfected with 70% ethanol and betadine. A midline incision was made at the C4–T1 area, and the skin and superficial muscles were retracted. Following C5–C6 and C6–C7 resection of the ligamentum flavum, removal of the periosteal under-surface of the C6 lamina was performed using a microhook. Then the compression material was implanted underneath the C6 lamina using microsurgical techniques (Figs. 1A to C). In the sham operated group the compression material was removed 30 s after implantation. Multilayer tissue closure was then performed. Decompressive surgery To test the potential use of this novel CSM model in discovering novel neuroprotective treatments complementary to surgical intervention, surgical decompression of the cervical spinal cord was attempted at 5 weeks post-surgery in the decompression group. In short, under anesthesia a midline incision was made over C4–T1 area, skin and superficial muscles were retracted and C5, C6 and C7 laminae were exposed. Using a microdrill, the upper half of C7 lamina and the lower half of C5 lamina were removed. In turn, the left half of C6 lamina was removed using the microdrill. Then, the soft tissue exposed underneath C6 lamina was divided using a microhook and microscissors at the left side edge around lateral mass. The remaining half of the C6 lamina was removed from the midline to lateral. While holding the remained half of C6 lamina at the left side with a fine forceps, the exposed soft tissue with bone forming film was divided from the dural sac by microhook from left side to right side. Finally, by cutting the right side of the C6 lamina, the C6 lamina and the compression formation underneath were removed. Successful decompression was confirmed by dural sac pulsation. There was no cerebrospinal fluid leakage.

Fig. 1. Intraoperative images showing the three-step process of material implantation. Following resection of C5–6 and C6–7 ligamentum flavum (A), the remaining tissue attached underneath the C6 lamina and the periosteum of the inner surface of C6 lamina was removed using a microhook (B). Then a piece of synthetic polyether material was slipped underneath the C6 lamina without the need for laminectomy (C).

Neurobehavioral assessments For behavioral testing, pre-training of rats started at 2 weeks before surgery in all behavioral tasks. All behavioral tests have been performed and analyzed “blinded” to the treatment groups. Automated gait analysis (CatWalk) Gait analysis was performed using the CatWalk system (Noldus Information Technology, Wageningen, Netherlands) described (Hamers et al., 2006; Koopmans et al., 2005). In short, the system consists of a horizontal glass plate and video capturing equipment placed underneath and connected to a PC. In our work, for correct analysis of the gait adaptations to the chronic compression, after standardization of the crossing speed, the following criteria concerning walkway crossing were used: (1) the rat needed to cross the walkway, without any interruption (2) a minimum of three correct crossings per animal

Table 1 Description of the experimental groups and experimental procedures. Groups

Material implantation

Surgical decompression

Neurobehavioral studies

MRI

Electrophysiology

BDA/EB injections

Histology/IHC

Sham Compression Decompression

+ + +

− − +

+ + −

+ + +

+ + −

+ + −

+ + −

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was required. Files were collected and analyzed using the CatWalk program, version 7.1. Individual digital prints were manually labeled by one observer blinded to groups. With the CatWalk, a vast variety of static and dynamic gait parameters can be measured. In the present study, we examined the following parameters, most of which have been studied in human CSM gait analysis: • • • • • • •

base of support (expressed in mm): distance between both hindpaws number of steps stride length stance phase duration % of 4-limb support swing speed relative paw placement (expressed in mm): distance between the placement of the ipsilateral fore- and hindpaws of the animal. When the hindpaw is placed upward compared with the forepaw, the value is negative. A positive value indicates that the hindpaw is placed backward compared with the forepaw.

Before surgery, animals were acclimated and trained to the walking apparatus following the method described by Gensel et al. (2006).

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pre-clinical system (Bruker BioSpec 70/30 USR). Rats, maintained at 1.8% isoflurane, were oriented in prone position within a linear volume resonator for RF transmission. A 30-mm diameter flat receiver coil was fixed in place directly above their cervical area. T1-weighted 2D-RARE images were acquired in sagittal planes within the region of material implantation with the following parameters: echo time: 16 ms, repetition time: 1000 ms, field-of-view: 30 mm, resolution: 150 × 150 × 1000-micron, number of excitations: 8, and acquisition time: 13 min. Respiratory gating was used during imaging. Based on mid-sagittal MR images, the compression ratio was calculated using the following formula (Fehlings and Tator, 1995): MRI compression ratio (%) = 1 − (2c / a + b) × 100%, where “c” is the anteroposterior canal diameter at the level of maximum compression, “a” is the anteroposterior canal diameter at the nearest normal level above the site of compression, and “b” is the anteroposterior canal diameter at the nearest normal level below the site of compression. The distances were calculated by a blinded observer using the Medical Image Processing, Analysis, and Visualization (MIPAV) software (http://mipav. cit.nih.gov). Electrophysiology

Hand grip strength test The grip strength test assesses motor function by recording the amount of force exerted by the forelimb (SDI Grip Strength System model DFM-10; San Diego Instruments, San Diego, CA). The grip strength test was performed in the compression and sham groups at first and second weeks post-surgery and biweekly thereafter as it is described (Lee et al., 2012). Assessment of mechanical allodynia by von Frey filament testing Cutaneous sensitivity to innocuous mechanical stimulation of both forepaws and hindpaws was assessed at first and second weeks postsurgery and biweekly thereafter in both compression and sham groups using a series of filaments of varying thickness (von Frey filaments) applied to the mid-plantar surface of forepaws and hindpaws, one paw at a time. We used 14 Touched-Test von Frey filaments, number 5–16 (North Coast Medical, Inc., CA, USA) with a regularly calibrated stiffness corresponding to 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10, 15, and 26 g. Probing was only performed when the animal's four paws were in contact with the floor. Each probe was applied to the foot until it was bent. A minimum of three withdrawals of the tested paw out of five filament applications was considered a positive response. Filaments were applied in ascending order, and the smallest filament that elicits a positive response was considered the threshold stimulus. Assessment of thermal hyperalgesia by the tail flick test The thermal nociceptive response was evaluated biweekly in all animals of the compression and sham-operated group by recording the latency to withdrawal of the tail in response to noxious skin heating. In short, the dorsal surface of the tail between 4 and 6 cm from the tip of the tail was exposed to a beam of light generated from an automated analgesia meter (IITC Life Science, Woodland Hills, CA). The timer was stopped when the animal flicked its tail away from the beam of light. Tail-flick latency was measured at 5-minute intervals until a stable baseline was obtained over 3 consecutive trials. The mean latency was used as a measure to indicate thermal hyperalgesia. MRI The extent of compression of the spinal cord was quantitatively evaluated in the compression group using MRI at 10 weeks postsurgery. For confirmation of surgical decompression of the cervical spinal cord, MRI was acquired in the decompression group just before (5 weeks post-surgery) and 5 weeks after decompression. The MRI was carried out at the STTARR facility in Toronto, using a 7 Tesla

Sensory-evoked potentials At 10 weeks post-surgery, sensory-evoked potentials (SEPs) were recorded from rats under isoflurane anesthesia. The rats were fixed in a stereotaxic holder in a prone position. After removing interlaminar ligaments between C1 and C2, two pairs of 1.0-mm ball electrodes were positioned extradurally over the spinal cord at C1 and C2. Two stainless steel needle electrodes were inserted into the forepaw. A constant current stimulus 0.1 ms in duration and 2.0 mA in intensity was applied at a rate of 5.7 Hz to the forepaw. At a bandwidth of 10–3000 Hz, a total of 2000 SEPs were averaged and replicated. SEP peak latency was measured from the start of the stimulus (S) to the peak of first negative peak (N1). The evoked potential amplitudes were measured as the voltage difference from the peak of the first positive peak (P1) to the peak of the first negative peak (N1). Hoffman reflex recording In our experiment, recording electrodes were placed two cm apart in the mid calf region and the posterior tibial nerve was stimulated in the popliteal fossa using a 0.1 ms duration square wave pulse at a frequency of 1 Hz. The recordings were filtered between 10 and 10,000 Hz. Recordings of the H-reflex typically consist of two EMG responses—an initial M wave and a later H-wave. The M-response is the result of the direct activation of the motor axons and does not involve the spinal circuits. The later H-reflex is a compound EMG response in the plantar muscle elicited by the synaptic activation of motoneurons by muscle afferents. The threshold for both the M and H waves was determined, and the Hmax/Mmax ratio was calculated. Corticospinal tract (CST) Labeling of the CST was performed by using biotinylated dextran amine (BDA) injected to the motor cortex (Guest et al., 2008) and by PKC-γ immunostaining (Karimi-Abdolrezaee et al., 2010). At 7 weeks post-surgery, under halothane anesthesia, rats were positioned in a stereotaxic frame and bilateral craniotomies were created using a surgical microdrill. Biotinylated dextran amine (BDA) (10%, 10,000 MW; Invitrogen) was injected bilaterally into the motor cortex (Guest et al., 2008). Injections performed at 5 sites (0.5 μl per site) in each side lateral to the bregma using the following coordinates (in reference to bregma): (1) 1.0 mm anterior and 2.5 mm lateral; (2) 0.5 mm anterior and 2.5 mm lateral; (3) 0.5 mm posterior and 2.5 mm lateral; (4) 1.0 mm posterior and 2.5 mm lateral; and (5) 1.5 mm posterior and 2.5 mm lateral. Injections were made at a depth of 1.2 mm from the surface of the cortex.

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Blood spinal cord barrier (BSCB) integrity To evaluate the BSCB integrity under the chronic cervical spinal cord compression animals from both compression and sham groups were injected with 100 μl of 4% Evans Blue (EB) [molecular weight (MW), 961 Da; Sigma Aldrich] at 10 weeks post-surgery one hour before the sacrifice. Tissue processing Animals were fixed by transcardial perfusion with 4% paraformaldehyde (Sigma Aldrich, St Louis, MO, USA) in PBS. Spinal cord centered at CE was harvested and post-fixed in 4% paraformaldehyde containing 10% sucrose overnight followed by PBS containing 20% sucrose for 48 h at 4 °C. Cords were then frozen in optimal cutting temperature (OCT) matrix and sectioned either every 20 or 40 μm. Histopathology Serial transverse spinal cord sections at every 20 μm until 2000 μm rostral and caudal to compression epicenter (CE) were prepared. To assess the effects of chronic compression on gray matter preservation as well as on scar tissue and cavity formation, tissue sections for each animal were systematically sampled every 120 μm over a distance of 4000 μm and were stained with the cellular stain hematoxylin–eosin (H&E) and myelin-selective pigment Luxol Fast Blue (LFB). Scar tissue was defined as tissue that exhibited a fibrous inconsistent tissue matrix. Tissue section areas were obtained using the Cavalieri method of Stereo Investigator (MBF Bioscience, Williston, VT, USA). The percentage of scar tissue and cavity area for each section was calculated using the following formula: % of scar tissue and cavity of tissue section = area of scar tissue and cavity of tissue section / total area of section. The percentage of the preserved gray matter and that of the white matter was calculated using the following formulas: % of preserved gray matter of tissue section = area of gray matter of tissue section / total area of tissue section. Immunohistochemistry For all immunohistochemical stainings, the blocking solution contained 5% nonfat milk, 1% bovine serum albumin, and 0.3% Triton X-100 in 0.1 M PBS. The frozen sections were air dried at room temperature for 10 min and then were washed with PBS for 10 min. For identification of motoneurons and vessels the following antibodies were used: mouse anti-choline acetyltransferase (ChAT, 1:200; Millipore, Temecula, CA) and monoclonal mouse anti-rat endothelial cell antibody 1 (RECA-1, 1:50; MCA970R, Serotec). BDA and PKC-γ 21 days following BDA injection animals were perfused, and the spinal cord segments were embedded and cut as described above. Seven transverse sections per animal at CE and 1, 3, and 5 mm caudal and rostral to CE were selected for immunohistochemical processing against BDA. For BDA visualization, sections were incubated with the following reagents: 0.3% hydrogen peroxide (H2O2) in absolute methanol for 30 min at 4 °C (using blocking solution as above), Vectastain AB (ABC Elite Kit, Vector Laboratories) in PBS containing 0.5% Triton X according to the manufacturer's instructions for 2 h, 0.05% diaminobenzidine, and 0.05% H2O2 in PBS for 5 min. Sections were mounted onto slides and air dried. Then, the slides were dehydrated through an alcohol series, cleared in xylene, and coverslipped. For PKC-γ immunostaining, complementary spinal cord sections were blocked and then incubated with mouse anti-PKC-γ antibody (1:200; Santa Cruz Biotechnology) overnight. After washes in PBS, the sections were incubated with fluorescent Alexa 568 goat anti-mouse secondary antibody for 1 h (1:400;

Invitrogen, Burlington, Canada). Then sections were slide mounted and coverslipped with Mowiol. Apoptosis & double labeling To examine apoptotic DNA degradation using the TUNEL assay we employed the ApopTag Fluorescein In Situ Apoptosis Detection Kit (#S7110, Chemicon International). The TUNEL assay was performed between the wash step after primary antibody and the incubation step with the secondary antibodies, following the manufacturer's instructions. As a negative control to assess the specificity of this procedure, selected sections were stained in the absence of TdT. In addition, to examine active cellular apoptosis immunofluorescence staining was performed with rabbit anti-cleaved caspase-3 (Asp175) antibody (1:300; Cell Signalling Technology, Inc., Beverly, MA). Following TUNEL assay sections were incubated overnight with one of the following cell-specific markers: mouse antineuronal nucleus (NeuN; 1:500; Chemicon International, Inc., Temecula, CA) or Olig2 (1:1000; Millipore). Moreover, following cleaved caspase-3 staining, the sections were incubated overnight with mouse anti-NeuN or mouse anti-adenomatous polyposis coli (APC; 1:100; Calbiochem, San Diego, CA) for oligodendrocytes. Sections were washed three times with PBS and incubated with fluorescent Alexa 488, 568 or 647 goat anti-mouse/rabbit (1:400; Invitrogen, Burlington, Canada) for 1 h. Then all sections were rinsed three times with PBS and coverslipped with Mowiol mounting media containing DAPI (Vector Laboratories, Inc., Burlingame, CA) to counter-stain the nuclei. As negative controls, the same procedure was performed without primary antibodies. Image processing and analysis Quantification of axonal density in the corticospinal tract To examine the integrity of main CST, we used immunostaining for PKC-γ, which specifically marks main CST in the dorsal column. For the assessment of PKC-γ in the CST, we photographed the dorsal columns of the spinal cord at 10 × magnification at 1, 3, and 5 mm rostral and caudal to CE and at CE. Using ImageJ Software (NIH), we measured the relative immunodensity of PKC-γ in a traced area within the dorsal columns (depicted as a circle in Fig. 8). Background intensity was deducted from the average gross intensity to correct for nonspecific reactions. Furthermore, we performed automatic thresholding for each image using ImageJ to determine the threshold for specific signal. After setting the threshold, immunodensity above this threshold was automatically calculated. Moreover, we used anterograde BDA labeling to complement the study of CST axonal degeneration under chronic compression. For this purpose, we undertook the same approach as described above for PKC-γ to measure the BDA integrated density within the same defined region of the dorsal columns (see Fig. 8D, circled region). Quantification of vessels and EB extravasation For vessel quantification we used sections immunostained with a monoclonal antibody specific against rat endothelial cell antigen. The counting of vessels was performed at 20 × magnification on 4 selected fields (ventral horn, dorsal horn, left and right lateral columns). Then, the obtained values were pooled and the number of vessels was calculated at 0.5, 1 and 3 mm caudal and rostral to CE. EB is widely used to measure vascular protein leakage. Increased vascular leakage causes the leakage of albumin out of blood vessels. Since EB binds to albumin in the circulation, vascular leakage can be evaluated by measuring the EB that has exuded from blood vessels. The primary BSCB breakdown, reflected by the leakage of fluorescent EB, was evaluated at 1, 3, and 5 mm rostral and caudal to CE and at CE. Images from spinal cord tissue sections from both experimental groups were captured together by a blind observer using the same gain and exposure on Leica microscope. Then, using ImageJ analysis

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system (NIH) we traced the entire spinal cord tissue areas. Furthermore, we performed automatic thresholding for each image using ImageJ to determine the threshold for specific signal. After setting the threshold, the immunodensity above the threshold was automatically calculated. We then divided the integrated density by the sample area to calculate the mean density per unit area. This calculation was performed to compensate for the different sizes of the spinal cords.

frame or were touching the inclusion lines were counted as the nuclei came into view while focusing through the z-axis. Settings for counting were: 1 / Section Sampling Factor (SSF) = 12; 1 / Area Sampling Factor (ASF) = 2.77 for TUNEL and cleaved caspase-3 positive cells and 27.77 for motoneurons; TSF = 0.67; guard zone = 3 μm.

Stereological methods For motoneuron, apoptotic neurons or oligodendrocyte quantification we used unbiased stereology counting. For unbiased stereology, it was essential that the entire 4 mm of tissue including the compression epicenter, beginning at a random starting point (chosen using the random number generator on the Stereology software), was sampled every twelfth section. The multilevel sampling design based on the Optical fractionator sampling method of the StereoInvestigator software, was used to estimate immunolabeled cell numbers. For motoneuron counting, the two anterior horns were traced and a systematic random sample set was generated with each grid measuring 200 × 200 mm and the Optical fractionator unbiased counting frame measuring 120 × 120 mm. For detecting apoptotic neurons and oligodendrocytes, the gray matter and the whole spinal cord tissue section were traced, respectively. Then, a systematic random sample set was generated with each grid measuring 420 × 420 mm and the Optical fractionator unbiased counting frame measuring 80 × 80 mm. The thickness of each section following processing was measured using the StereoInvestigator system XYZ stage controller with the average thickness being reduced to 80% of the initial thickness. Using the Acquire SRS Image Series Workflow, images from each unbiased counting frame were acquired with the use of a computer driven motorized stage to shift through the region of interest. For all regions, cells were counted using the Optical fractionator workflow probe. Total cell number was counted using DAPI to identify cell nuclei, and was counted separately due to differences in each immunofluorescence profile. Immunostained profiles that fell within the counting

To obtain total protein, 1 cm long spinal cord samples centered at the CE site from compression and sham rats (n = 4/group) were individually homogenized in 5 mM Tris–HCI (4 mM EDTA, pH 7.4, containing 1 μM pepstatin, 100 μM leupeptin, 100 μM phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin) and were cleared by centrifugation at 10,000 g for 10 min at 4 °C. Protein concentrations were determined by the Lowry method (Bio-Rad Laboratories, Hercules, CA). Approximately 30 μg of protein was run on a discontinuous SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat milk in Tris buffered saline with 0.05% tween-20 (TTBS) for 1 h at room temperature followed by the application of primary antibodies. They were then incubated with the following primary antibodies: (1) rabbit anti-Iba-1 (1:1000, Wako); (2) rabbit anti-Bcl-xl (1:1000, Santa-Cruz); (3) rabbit antiBad (1:1000, Santa Cruz) and (4) rabbit anti-Bax (1:1000, Santa Cruz) in blocking solution overnight at 4 °C. Membranes were then rinsed for 10 min in TTBS. Secondary horseradish peroxidase antibodies (Sigma) were incubated at room temperature for 1 h in blocking solution. Protein bands were then visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences Inc, Buckinghamshire, UK) and exposed to film. To quantify the amount of protein, the bands were first determined to be within the linear range of the radiographic film, and optical densities were then determined by measuring the integrated optical density across the band using Gel Pro analysis software (Media Cybernetics, Silver Spring, MD). Densitometric values were normalized to those of β-actin (1:400; Sigma).

Western blot analysis

Fig. 2. Chronic compression reproduces the main human CSM neuropathological features. (A) H–E & LFB stained transverse sections coming from compression epicenter and from sham-operated animals (scale bar: 50 μm). (B) Overall the extent of preserved gray matter area was found to be significantly decreased in the compression (C) group compared to the sham (S) group. (C) Scar tissue and cavity areas were found to be statistically significantly increased in the compression group. No scar tissue was found in the sham-operated group [n = 6 in compression group and 5 in sham group, two-way ANOVA with Bonferroni post hoc; * p b 0.05, ** p b 0.001]. Data are presented as mean ± SEM.

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Statistical analysis

MRI

Statistical analysis was performed using SigmaStat software (SPSS). Two-way ANOVA, followed by post hoc pair wise multiple comparisons and t-tests were used accordingly. The number of animals per group is included in the Results section and figure legends. Data are presented as mean values ± SEM and p b 0.05 was considered significant.

As depicted in Fig. 3, MRI studies revealed postero-anterior cervical spinal compression at the C6 level in animals of the compression group. Quantitative assessment of the extent of compression revealed a compression ratio of 48.4 ± 4.96 at 10 weeks post-surgery in CSM rats (n = 10). Moreover, comparison of MRI and histologic studies of the same animals revealed that the low T1 intramedullary signal changes reflected cavity and glial scar formation in the spinal cord. Finally, MRI studies showed that surgical decompression of the spinal cord was successfully performed after C6 laminectomy, and C5 and C7 hemi-laminectomies in rats of the decompression group (n = 5) (Figs. 3A to D).

Results Chronic compression resulted in a significant loss of gray matter and increased scar tissue area Rats with compressive lesions displayed cystic cavitation, scar tissue, central gray and white matter degeneration, atrophy of the anterior horn of gray matter and anterior horn cell loss on hematoxylin–eosin (HE) and Luxol Fast Blue (LFB) sections. Moreover, significant loss of gray matter area and increased scar tissue and cavity area (two-way ANOVA, p b 0.001 for each) was observed over a rostro-caudal distance of 4 mm centered on the compression epicenter (CE) and was found in the CSM (n = 6) group compared to the sham (n = 4) group (Figs. 2A to C).

CSM rats demonstrated a broad-based, arrhythmic and spastic gait pattern In the present study, the distance between both hindpaws was analyzed to assess the hindlimb base of support (BOS). As shown in Fig. 4A hindlimb BOS increased progressively after the 2nd week post-surgery and remained almost constant thereafter in animals of the compression group. However, in the sham group, the hindlimb BOS remained unchanged during the course of the study. The large

Fig. 3. Postero-anterior compression of the cervical spinal cord at C6 level. (A) T1 weighted mid-sagittal MRI performed on compression group animals at 10 weeks post-surgery shows postero-anterior cervical spinal cord compression at C6 level with laminae C7 and C5 being intact. (B) Bar graph illustrating the quantitation of the extent of compression in CSM rats at 10 weeks post-surgery. (C) Comparison between the T1 weighted mid-sagittal MRI (left) and histopathology studies (right) derived from the same animal revealed that low T1 intramedullary signal changes in MRI (circled box) mirror cavity formation and glial scar tissue in the spinal cord. (D) MRI performed on the animals of the decompression group before and after decompressive surgery confirmed the successful surgical decompression of the cervical spinal cord. Data are presented as mean ± SEM.

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Fig. 4. Progressive compression of the cervical spinal cord in CSM rats is associated with progressive gait dysfunction. (A) There was an overall difference in hindlimb base of support (BOS) between the compression (C) and the sham (S) operated groups (two-way ANOVA, p b 0.001). BOS was not significantly altered in sham-operated animals over the course of the study. The hindlimb BOS in animals of the compression group was significant increased at 4, 6, 8 and 10 weeks post-surgery as compared to the sham group. Moreover, the hindlimb BOS of the compression group at 10 weeks post-surgery was significantly higher than that at 4 weeks post-surgery. (Bonferroni post hoc, p = 0.039 for 4 weeks vs 10 weeks). (B) A comparison of the walking track hindlimb footprints for sham and CSM was made. Sham operated animals exhibit a normal, small distance between the placements of the hindpaws during walking. Animals with chronic progressive cervical cord compression demonstrated a large distance between the placements of the hindpaws which denotes increased BOS at 5 weeks post-surgery. (C) The number of steps needed to cover the walkway was calculated and plotted for each group. There was an overall difference in the number of steps between the compression and the sham groups (two-way ANOVA, p b 0.001). The number of steps was not significantly altered in sham group over the course of the study. At 4, 6, 8 and 10 weeks post-surgery, rats of the compression group needed more steps to cover the walkway compared to sham-operated animals. Moreover, at 10 weeks post-surgery, CSM rats needed more steps to cover the walkway compared to at 4 weeks post-surgery. (Bonferroni post hoc, p = 0.021 for 4 weeks vs 10 weeks). (D) There was an overall difference in forelimb stride length (SL) between the compression and sham groups (two-way ANOVA, p b 0.001). SL was not significantly altered in sham-operated animals over the course of the study. However, the SL was significantly decreased in CSM animals compared to sham at 4, 6, 8 and 10 weeks post-surgery. Moreover, there was a significant difference between the 6th and 10th week's forelimb SLs of the compression group (Bonferroni post hoc, p = 0.036 for 6 vs. 10 weeks). (E) Although the hindlimb SL of sham-operated animals remained unchanged during the course of the study, in CSM animals it was found to progressively decrease over time. There was a significant difference between the 6th and 10th week's hindlimb SLs of the compression group (Bonferroni post hoc, p = 0.027 for 6 vs. 10 weeks). (F) Raw data from animals on the walkway in false color. Sham animals exhibiting a normal stepping pattern with a 1:1 ratio of fore- to hindlimb steps and overlapping fore- and hindlimb placements. Stride length is the distance between two consecutive steps with the same limb. Walking pattern of CSM animal demonstrated decreased stride length, loss of inter- and intralimb coordination and an increased ratio of fore- to hindlimb ratio reflecting spinal cord dysfunction. (G) There was an overall difference in forelimb stance phase duration between the compression and the sham groups (two-way ANOVA, p b 0.001). Forelimb stance phase was initially found to be significantly decreased at 4 weeks post-surgery in the compression group and continued to be decreased thereafter. At 6 weeks post-surgery the forelimb stance phase of the compression group was significantly lower than that observed at 10 weeks post-surgery (Bonferonni post-hoc, p = 0.032). (H) Although the hindlimb stance phase of sham-operated animals remained unchanged during the course of the study, in CSM animals it was found to progressively decrease over time There was a significant difference between the 6th and 10th week's hindlimb stance phase durations of the compression group (Bonferroni post hoc, p = 0.019 for 6 vs. 10 weeks). (I) The % of 4-limb was found to progressively increase in the compression group compared to sham animals [n = 10 in compression group, n = 6 in sham group, two-way ANOVA with Bonferroni post hoc; * p b 0.05, ** p b 0.001]. Data are presented as mean ± SEM.

distance between right and left hindpaw placements in CSM animals compared to sham operated animals is depicted in Fig. 4B. Moreover, animals in the compression group demonstrated progressively decreased stride length in both fore- and hindlimbs, starting at 4 weeks post-surgery. Consequently, these rats showed a progressive increase in the number of steps required to cover the walkway compared to sham group (Figs. 4A to E). In addition the relative paw placement in compression and sham groups was evaluated. The CatWalk

parameter “relative paw placement” defines the distance between the placement of the ipsilateral fore- and hindpaws. While sham operated animals place their hindpaws at or close to the previous position of the forepaw, this ability was progressively lost during the progressive compression of the cervical spinal cord in CSM rats as indicated by the large distance between paw placement positions (Supplementary Figs. 2A & B). Besides the static parameters, dynamic parameters such as stance phase duration, percentage of 4-limb support and

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swing speed were affected in CSM animals. Specifically, stance phase duration progressively increased over time in the fore- and hindlimbs of compression group animals. Furthermore, the percentage of 4-limb support during walking was increased over time in the compression group while it remained constantly low in the sham group (Figs. 4F to I). On the other hand, swing speed progressively declined in both fore- and hindlimbs of the compression group compared with sham animals (Supplementary Figs. 2C & D). Progressive forelimb weakness in compression group Although sham-operated animals showed no significant change in their grip strength over the course of the study, rats in the compression group experienced significant reductions in grip strength at 4, 6, 8 and 10 weeks post-surgery. Finally, forelimb dysfunction in CSM rats was more prominent at 10 vs 4 weeks post-surgery (Bonferroni post-hoc, p = 0.039) (Fig. 5A). Sensory changes at and below the level of compression Forepaw sensitivity to mechanical stimulation was not significantly altered in sham-operated animals (n = 6) over the course of the

study. There was no difference between the compression (n = 10) and sham group at the first post-operative week (Bonferroni post-hoc, p = 0.76, for sham vs compression groups). However, the compression group demonstrated significant progressive increases in sensitivity over a 9 week period (Bonferroni post-hoc, p b 0.001 for each time point). Hindpaw mechanical allodynia and tail flick tests in sham (n = 6) and compression (n = 10) groups revealed the development of sensory changes below the compression level; however, this occurred in a delayed fashion compared to the forepaw (Figs. 5B to D).

Chronic compression resulted in significant decline in forelimb sensory evoked potential (SEP) amplitude and an increase in hindlimb H-reflex The maximal H-reflex was expressed as a ratio of the Hmax/Mmax. At 10 weeks post-surgery the mean value obtained for maximal H-reflexes in the compression group was significantly higher than in sham rats (t-test, p b 0.05). SEPs were analyzed by peak amplitude measurements from the first positive peak to the first negative peak. At 10 weeks post-surgery peak amplitude was significantly lower in CSM rats compared to shams (t-test, p b 0.05) (Figs. 6A to D).

Fig. 5. Chronic and progressive compression of the cervical spinal cord leads to progressive forelimb weakness and neuropathic pain at and below the compression level. (A) Grip strength test power. There was no difference between the sham (S) and compression (C) groups at 1 and 2 weeks post-surgery (Bonferroni post-hoc, p = 1 and p = 0.94, for sham group vs. compression group at 1 and 2 weeks, respectively). There was no difference between the sham and compression groups at 1 and 2 weeks post-surgery (Bonferroni post-hoc, p = 1 and p = 0.94, for sham vs. compression group at 1 and 2 weeks, respectively), whereas grip power was significantly lower in the compression compared to sham group at 4, 6, 8 and 10 weeks post-surgery (Bonferroni post-hoc, p b 0.05, for sham group vs. compression group at 4 weeks post surgery and p b 0.001 for 6, 8 and 10 weeks, respectively). (C) Both the fore- and hindpaws of the rats in the compression group demonstrated significantly decreased withdrawal thresholds compared to the sham group (two way ANOVA, p b 0.001 for each). Moreover, there was a significant difference between the 4 week and 10 week withdrawal thresholds of the forepaws in the compression group (Bonferoni post-hoc, p b 0.05). (D) Tail flick test for thermal hyperalgesia. Graph illustrating the changes in tail withdrawal latency to thermal noxious stimuli following chronic compression. Animals of the compression group started demonstrating significantly decreased withdrawal latency at 4 weeks post-surgery compared to the sham group. Moreover the withdrawal latency of the compression group at 4 weeks post-surgery was significantly greater compared to that of 10 weeks post-surgery (Bonferroni post-hoc, p b 0.05). [n = 10 in compression group and 6 in sham group, two-way ANOVA with Bonferroni post hoc; * p b 0.05, ** p b 0.001]. Data are presented as mean ± SEM.

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Fig. 6. H-reflex and sensory-evoked potentials. (A) Representative waveforms of H-reflex are shown for CSM and sham animals. (B) In our rats we tested maximal plantar H-reflex/ maximal plantar M-response (HMAX/MMAX) ratios to determine the excitability of the H-reflex. Analysis of the recordings revealed that the mean value obtained for maximal H-reflexes in sham (S) operated animals was significantly lower than those that underwent compression (C). (C) Representative waveforms of sensory-evoked potential are shown for sham and compression group. (D) Analysis of sensory-evoked potentials revealed that peak amplitude was significantly lower in the compression (C) group compared with the sham (S) group [n = 5 in sham group and 6 in compression group, t-test; * p b 0.05].

Chronic compression led to motoneuronal cell loss

Compromise of the cervical spinal cord microvasculature

Unbiased stereology was used to count the total number of motoneurons (ChAT-positive cells) within a 5 mm length of spinal cord tissue including the CE in the compression group (n = 5) and within a complementary area of spinal cord tissue in the sham group (n = 3). As depicted in Fig. 7B, the estimated number of total motoneurons was found to be significantly decreased in the compression group compared to the sham group (t-test, p b 0.001).

To quantify the effects of chronic compression on the number of vessels, we performed immunostaining with RECA-1 in both compression (n = 6) and sham (n = 4) groups. Decreased capillary density was found in the gray and white matters in the compressed spinal cords compared to sham. Moreover at 10 weeks post-surgery, the EB intensity in the total spinal cord section area of the compression group (n = 5) was found to be increased by a statistically significant amount compared to sham animals (n = 3) (two-way ANOVA, p = 0.008). These results are indicative of BSCB disruption under chronic progressive compression of the cervical spinal cord (Figs. 9A to D). For evaluating the macrophage/microglia activation in the chronic compressed cervical spinal cord, the Iba-1 protein expression was measured using immunoblotting (n = 3/per group). Fig. 9E demonstrates representative Iba-1 Western blot bands for non-compressed control (sham) and chronic compressed animals. Western blot analysis of Iba-1 protein expression at 10 weeks post-material implantation showed that there was a statistically significant difference in the relative densities between the sham and the chronic compressed animals (n = 3 per group; p = 0.048) (Fig. 9D).

Corticospinal tract (CST) degeneration We assessed the integrity of the main CST using immunostaining for PKC-γ, which specifically marks the main CST in the base of the dorsal columns of uncompressed rats (Karimi-Abdolrezaee et al., 2010). At 10 weeks post-surgery, we found dramatically decreased PKC-γ in the dorsal columns of CSM spinal cord sections (n = 5) at all examined distances caudal, and at 1 and 3 mm rostral, to CE compared to the uncompressed spinal cords (n = 3). Disrupted CST fibers frequently appeared as swollen structures indicating dystrophic terminals (white arrow). Similarly, in complementary sections, decreased BDA-integrated density was found at the same examined distances caudal and rostral to CE. These results suggest that under chronic progressive compression of the cervical spinal cord, CST caudal axons undergo Wallerian degeneration, whereas the axons in the rostral segment are subjected to retrograde axonal degeneration or “die-back” (Figs. 8A to E).

Chronic compression activates the pro-apoptotic pathways and induces neural and oligodendrocyte apoptosis To examine the activation of pro-apoptotic pathways under chronic cervical spinal cord compression, the expression pattern of Bcl-xl,

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Fig. 7. Choline Acetyltransferase (ChAT) positive cells were significantly reduced in the compression group. (A) Confocal images stained with ChAT in red coming from the anterior horn of the gray matter of CSM and sham spinal cord sections. A significant decrease in the number of motoneurons was found in the compression group compared to the sham group. Interestingly, the preserved motoneurons in the CSM animals were found to be smaller in size as well. (B) Chronic progressive compression of the cervical spinal cord significantly decreased the number of motoneurons within a 5 mm spinal cord tissue area including the compression epicenter, as measured by unbiased stereological counting [t-test, * p b 0.001; n = 5 in sham (S) group, 6 in compression (C) group]. Data are presented as mean ± SEM. Scale bar: 20 μm.

Bax, and Bad proteins was evaluated using Western blot analysis in CSM and sham animals (n = 3/group). Fig. 10A shows representative Bcl-xl and Bax Western blot bands for non-compressed controls (sham) and CSM animals in addition to semi-quantitative densitometry results (Figs. 10B & C). There was a statistically significant difference in the Bcl-xl and Bax relative densities between the sham and the compression groups (p = 0.048 for Bcl-xl and p = 0.044 for Bax; t-test, n = 3 per group). In contrast there was no difference in the Bad relative densities between the sham and the compression groups (data not shown). Unbiased stereology was used to count the total number of TUNEL positive cells within a 5 mm length of spinal cord tissue including the CE in the compression group (n = 5) and within a complementary area of spinal cord tissue in the sham group (n = 3). The total number of TUNEL positive cells was found to be significantly increased in the compression group compared to the sham group (t-test, p b 0.001). Quantitation of cells double-labeled with NeuN and TUNEL showed that many of these apoptotic cells were neurons. Double-labeling also demonstrated the presence of oligodendrocytes that were positive for TUNEL (Figs. 11A to D). Moreover, quantitation of cells double-labeled with NeuN and cleaved-caspase-3 revealed that the number of neurons undergoing apoptosis was significantly increased within a 5 mm length of spinal cord tissue area including the CE in the compression group (n = 5) compared to the sham group (n = 3) (t-test, p b 0.001). Finally within the same tissue area, cleaved-caspase 3 was also significantly increased in CC1 positive cells in the compression group compared to the sham group (t-test, p b 0.001), providing further evidence of oligodendroglial apoptosis in the CSM model (Figs. 12A to D).

Discussion In this study we characterized a novel rat model of chronic progressive cervical spinal cord compression that reflects the spatial and the temporal profiles of human disease. MRI confirmed the progressive compression as well as the feasibility of surgical decompression. This chronic progressive cord compression resulted in significant gait pattern and forelimb dysfunction similar to human CSM. Furthermore this model has facilitated the examination of microvasculature disruption, neural apoptosis, anterior horn cell/corticospinal tract degeneration, neuropathic pain and spasticity. Modeling the main pathophysiological mechanisms of CSM There are three important proposed pathophysiological factors in the development of CSM: (1) static mechanical; (2) dynamic mechanical factors and (3) spinal cord ischemia (Young, 2000). Mechanical factors The static mechanical factors include all causes which result in the reduction of spinal canal diameter and spinal cord compression (i.e. congenital spinal canal stenosis, disc herniation, osteophytes, hypertrophy of ligamentum flavum or posterior longitudinal ligament). The dynamic factors are abnormal forces such as repetitive trauma to the spinal cord caused by pressure against existing mechanical compression during flexion and extension of the cervical spine. The dynamic mechanical factors aggravate spinal cord damage precipitated by direct mechanical static compression. In our model the lack of laminectomy during the material implantation secures the same range of motion in

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Fig. 8. Chronic progressive compression of the cervical spinal cord causes degeneration of the corticospinal tract (CST). (A) Intense staining of PKC-γ in transverse sections of spinal cords from sham-operated animals at C6 level shows normal localization of CST in the ventral part of the dorsal columns (circled box) [10× magnification (left) and 20× magnification (right)]. At 10 weeks post-surgery, at rostral and caudal distances to compression epicenter (data shown for 3000 μm caudal), PKC-γ immunoreactivity in the ventral part of the dorsal columns (circled box) in rats of the compression group was significantly diminished, suggesting degeneration of CST [10× magnification (left) and 20× magnification (right)]. Disrupted CST fibers frequently appeared as swollen structures indicating dystrophic terminals at higher magnification images (arrow). (B) Quantification of the circled areas depicted in A at various distances in both experimental groups demonstrated a significant decrease in PKC-γ immunoreactivity in the compression (C) group compared to sham (S) group at 1000, 3000 and 5000 μm caudal and 1000, 3000 μm rostral to the compression epicenter. (C) Longitudinal sections of rat spinal cords coming from compression and sham groups were immunostained with PKC-γ. Photomicrographs were captured in the rostral–caudal orientation from left to right. Degeneration of CST is depicted in the compression group. Moreover, more extensive degeneration of the main CST is revealed in caudal rather than rostral areas to compression epicenter. (D) BDA-labeled CST is demonstrated in dorsal columns of the spinal cords of sham operated group in both low (left) and high (right) magnifications after bilateral injections of BDA into the sensorimotor cortex. In contrast, dramatic decrease of BDA-labeling in the dorsal columns of rats of the compression group is depicted at 3000 μm caudal to compression epicenter in low (right) and high (left) magnification images after bilateral BDA injection into the sensorimotor cortex further suggesting degeneration of CST. (E) Quantification of the depicted areas in circled boxes (D) revealed a significant decrease in BDA density in the compression group compared to sham group at all examined caudal distances to the compression epicenter. Moreover, a statistically significant decrease in BDA density is revealed at 1000 and 3000 μm rostral to the compression epicenter. [n = 3 in sham group, n = 6 in compression group; two-way ANOVA with Bonferroni post hoc; * p b 0.05, ** p b 0.001]. Data are presented as mean ± SEM. Scale bars: 100 μm.

the cervical spine before and after the surgery. Thus, in addition to static mechanical compression, dynamic repetitive trauma on the cord is also simulated in this model. Ischemia To date, indirect experimental (Gooding, 1974; Taylor, 1964; Wilson et al., 1969) and postmortem studies (Baron and Young, 2007) in CSM patients demonstrating abnormal histological findings, such as spinal cord necrosis and gray matter cavitations, have indicated that vascular mechanisms may be involved in the pathophysiology of CSM. Furthermore, the region of the spinal cord most affected by CSM (levels C5 to C7) is also the area with a vulnerable vascular supply (Baron and Young, 2007; Ferguson and Caplan, 1985; Firooznia et al., 1985; Yue et al., 2001). It has been hypothesized that one common factor with reference to mechanical distortion of spinal cord with anterior or posterior compression is flattening or widening of the spinal

cord, which may stretch the intrinsic transverse vessels or terminal branches of the anterior spinal artery (Emery, 2001). However, direct evidence describing the fate of spinal cord microvasculature under chronic mechanical compression is lacking. Our results demonstrate, for the first time, that chronic compression of the spinal cord causes flattening, stretching and consequent loss of vessels. Modeling the clinical features of CSM The insidious and delayed onset of symptoms and progression of deficits in our CSM animal model resembles the chronic and progressive nature of the human disease. Gait impairment is one of the cardinal clinical features of CSM in humans (Gorter, 1976; Lunsford et al., 1980). The typical human CSM gait, manifests as a spastic gait pattern and is qualitatively described as broad-based, ataxic, hesitant, and jerky, compared with the smooth, rhythmic, normal gait

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(McCormick et al., 2003). Specifically, this typical human CSM gait is quantitatively characterized by increased base of support, stance phase duration, and number of steps with decreased walking speed, stride length, and % single stance phase duration (Kuhtz-Buschbeck et al., 1999; Maezawa et al., 2001; Suzuki et al., 2002). Interestingly, in our study, gait analysis showed that CSM rats demonstrate progressive increases in base of support and loss of coordinated, reciprocal hindlimb

placement, which mirrors human CSM gait deficits. These results are further supported by changes in the H-reflex, a neurophysiological approach to assess a lower limb-derived monosynaptic spinal cord reflex arc which can be used to objectively assess spasticity in humans (Okamoto et al., 1980) and in rodents (Lee et al., 2005, 2009). Our data, indicate that with chronic progressive cervical cord compression, the H/M ratio increases at baseline and with high frequency stimulation.

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Fig. 10. Chronic cervical spinal cord compression induces pro-apoptotic pathway activation. (A) Representative images of Western blot bands. (B) At 10 weeks post material implantation, densitometry analysis showed that the expression levels of the Bcl-xl protein were statistically significant increased in the compression (C) group compared to the sham (S) operated group (n = 3 per group; t-test, p = 0.048). (C) Finally, as the Western blotting analysis showed, the expression pattern of the Bax protein at 10 weeks post material implantation was statistically significantly increased in the CSM animals compared to controls (n = 3 per group; t-test, p = 0.044; variance data transformation was performed). Data are presented as mean ± SEM.

These data fit with human results and corroborate lower extremity spasticity. CSM patients often experience upper extremity numbness and loss of fine motor control of the hands (Gorter, 1976; Mattei et al., 2011). Our CSM rats showed progressive forelimb weakness and neuropathic pain over the course of the study. Interestingly, the hand-grip strength test of CSM rats remained almost constant during the last weeks of the experiment suggesting possible endogenous rewiring mechanisms into the cervical enlargement regarding hand grasping. Finally, sensory changes were noticed in the tail and hindlimbs in a more delayed fashion.

This novel CSM model is MRI compatible and allows for surgical decompression To date, the challenges in interpreting the MRI changes in human CSM reflect the lack of an MRI-compatible animal model of this condition, which would allow precise correlation between the MRI signal changes and the corresponding histopathology. For example, while it is recognized that T1 signal changes are associated with a poor prognosis in CSM (Fernandez de Rota et al., 2007; Morio et al., 2001), there is little evidence related to the histopathological interpretation of these signal changes. In our study, we quantified the posterior–anterior compression of the spinal cord at C6 level using MRI. Moreover by comparing MRI with histopathological findings from the same animals, we found that the low T1 intramedullary signal changes reflect cavity and glial scar formation in the cervical spinal cord. The high resolution images and lack of material-related artifact make this model MRI-compatible. Interestingly, the MRI studies also confirmed that surgical decompression could be successfully undertaken post implantation. This is of translational relevance since it allows one to examine regenerative or reparative strategies, which are complementary to surgical decompression, which is in wide clinical use. To the best of our knowledge this model is the first one which satisfies this condition.

Histopathological correlations with the human disease Chronic compression of the human cervical spinal cord is associated with cystic cavitation, anterior horn atrophy as well as anterior horn cell loss in the gray matter (Baptiste and Fehlings, 2006; Fehlings and Skaf, 1998; McCormack and Weinstein, 1996; Swagerty, 1994; Yu et al., 1988). With regard to white matter, ascending and descending degeneration of the pyramidal tracts above and below the compression site are prominent (Baptiste and Fehlings, 2006; Fehlings and Skaf, 1998; McCormack and Weinstein, 1996; Swagerty, 1994; Yu et al., 1988). Indeed, spastic paraparesis manifested in CSM is considered to be, in part, mediated by the degeneration of the main CST. Our results demonstrated that the progressive chronic compression of the cervical spinal cord induced by our model leads to similar histopathological features as described in humans. Disruption of the BSCB and inflammation in CSM The spinal cord, like the central nervous system in general, is a relatively immunologically privileged zone. As an interface between the spinal cord and the periphery, the blood spinal cord barrier (BSCB) constitutes a physical/biochemical barrier between the CNS and systemic circulation and protects the microenvironment of the spinal cord. The BSCB is known to be destroyed in peripheral nervous system injury (Echeverry et al., 2011) and acute traumatic spinal cord injury(Beattie and Manley, 2011), but whether this is true in CSM was not known (Beattie and Manley, 2011). In the present study we showed, for the first time, that the BSCB is disrupted with slowly progressive cervical spinal cord compression. Moreover, we found an increased macrophage/microglia expression suggesting that the BSCB disruption promotes the inflammatory infiltration and/or exaggerates the existing neuroinflammation in CSM. The enhanced cross talk between the peripheral immune system and the spinal cord microenvironment, which occurs in CSM through an impaired BSCB, may have significant mechanistic implications that contribute to the progression of neural degeneration in CSM.

Fig. 9. (A) Middle panels: Low magnification (10×) images of transverse spinal cord sections stained with RECA-1 (red) and DAPI (blue). Significant reduction in the number of vessels in the gray and white matters is demonstrated in the CSM section when compared to sham (scale bar: 100 μm). The white boxed areas (lamina8 to lamina9) correspond to the confocal images shown in the left panels. The yellow circled areas (central canal-intermediate zone of the gray matter) correspond to the higher magnification (20×) images shown in the right panels. (B) Low magnification (10×) images of transverse spinal cord sections coming from sham and CSM animals after Evans Blue (EB) injection. EB (red) extravasation into the spinal cord parenchyma of CSM animal is more prominent in the gray rather than the white matter. No EB extravasation was found in the normal spinal cord (scale bar: 50 μm). (C) Bar graph depicting RECA-1 positive cell counts 10 weeks after the material implantation. The chronic compression led to a significant decrease in capillary density in CSM animals (n = 6) as compared with the sham group (n = 5). (D) Quantitation of EB autofluorescence intensity revealed that there was a statistically significant increase in extravasation of EB in rats in the compression (C, n = 5) group compared to the sham (S, n = 3) group. Specifically, there was a statistically significant difference in the EB autofluorescence intensity between the compression and sham groups at CE and at 1, 3 and 5 mm caudal to compression epicenter (Bonferroni post hoc, p b 0.001 for 1 and 3 mm and p b 0.05 for each examined distance). EB intensity was also significantly increased at CE compression epicenter and at 1 and 3 mm rostral to CE compared to sham spinal cord [two-way ANOVA with Bonferroni post hoc; * p b 0.05, ** p b 0.001]. (E) Chronic cervical spinal cord compression induces macrophage/microglia activation. Microglial/ macrophage activation was determined with Iba-1 expression at 10 weeks post surgery with Western blotting. There was a difference in Iba-1 protein expression between sham and CSM animals (n = 3 per group; t-test, p = 0.048). Data are presented as mean ± SEM.

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Fig. 11. Apoptotic neurons and oligodendrocytes in the compressed cervical cord. Representative micrograph of axial sections derived from CSM and sham-operated animals. (A) Double immunohistochemistry with TUNEL (green) and anti-NeuN (red) antibodies revealed TUNEL positive neurons in the CSM spinal cord sections in contrast with normal spinal cord sections. (B) Olig2 (fire red) labeling for oligodendrocytes and TUNEL (green) labeling demonstrated co-localization for TUNEL positive nuclei and Olig2 (merge, yellow), indicating DNA fragmentation of oligodendrocytes in CSM spinal cord tissue compared to spinal cord tissue derived from sham-operated animals (scale bar: 10 μm). (C & D) Chronic progressive compression of the cervical spinal cord significantly increased neuronal and oligodendrocytic apoptosis, as measured by unbiased stereological counting [t-test, * p b 0.001; n = 5 in sham (S) group, 6 in compression (C) group]. Data are presented as mean ± SEM.

Apoptosis In the present study, we show activation of the pro-apoptotic pathways in CSM. Moreover, we demonstrate co-localization of TUNEL positive nuclei with NeuN in addition to Olig2, thereby demonstrating apoptotic neuronal and oligodendroglial cell death in the cords of CSM rats. Moreover it was demonstrated that, after a 10 week compression period, neurons and oligodendrocytes express cleaved caspase-3 and thereby undergo apoptosis. Collectively, these findings are in agreement with previous reports, including studies from our own laboratory, showing that TUNEL positive neurons and oligodendrocytes and caspase-3 activation were observed at the compression site of the cervical spinal cord in 5- and 6-month-old twy/twy mice (Takenouchi et al., 2008; Yamaura et al., 2002; Yu et al., 2009; Yu et al., 2011). Demyelination and TUNEL positive cells have also been observed in the

chronically compressed post-mortem spinal cord of a deceased patient with OPLL (Yamaura et al., 2002). We believe that apoptosis in both neurons and oligodendrocytes is a crucial event which may contribute to progressive neural degeneration, demyelination and Wallerian degeneration, and thereby to the progressive nature of CSM. Our data add to the evidence that apoptosis in neurons and oligodendrocytes contributes to the pathobiology of spinal cord degeneration in CSM. Conclusion In conclusion the novel rat CSM model, described in the current paper, produces neurological deficits and neuropathological features accurately mimicking the human condition, is MRI compatible and, importantly, allows for surgical decompression. Hence it has the potential to identify the molecular mechanisms which lead to the progression of

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Fig. 12. Chronic progressive compression of the cervical spinal cord induces the caspase-3 apoptotic signaling cascade in both neurons and oligodendrocytes. (A) Double-labeling immunofluorescence with anti-cleaved caspase-3 and anti-NeuN antibodies was used to examine ongoing neuronal apoptosis in the compression and sham-operated groups. NeuN (red) labeling for neurons and cleaved caspase-3 (green) labeling for active caspase-3 demonstrated co-localization for cleaved caspase-3 and NeuN (merge, yellow), indicating neuronal caspase-3 activation in chronically compressed spinal cord tissue. No double-labeling was identified in spinal cord tissue derived from sham-operated animals (scale bar: 20 μm). (B) CC1 (red) labeling for oligodendrocytes and cleaved caspase-3 (green) labeling for active caspase-3 demonstrated co-localization for cleaved caspase-3 and CC1 (merge, yellow), indicating caspase-3 activation in oligodendrocytes in CSM spinal cord tissue. No double-labeling was identified in spinal cord tissue derived from sham-operated animals. Images were obtained 3 mm caudal to the compression epicenter in the anterior horn of a transverse spinal cord section derived from CSM modeling animals and sham. (C & D) Chronic progressive compression of the cervical spinal cord significantly increased the ongoing neuronal and oligodendrocytic apoptoses, as measured by unbiased stereological counting [t-test, * p b 0.001; n = 5 in sham (S) group, 6 in compression (C) group]. Data are presented as mean ± SEM.

this unique disease as well as to facilitate the discovery of novel clinically-relevant therapeutic targets for translation to human patients. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.nbd.2013.02.013.

acknowledge James W. Austin for sharing his research experience. This work was supported by the CIHR and the Gerald and Tootsie Halbert Chair in Neural Repair and Regeneration (MGF). We are also grateful for the financial support from the North America Spine Society (NASS). SKK was supported by a Synthes Spine Fellowship.

Acknowledgment The authors would like to acknowledge Warren Foltz at the STTARR facility for assistance with MR imaging and Behzad Azad for assistance with the animal care. Moreover the authors would like to

References Baptiste, D.C., Fehlings, M.G., 2006. Pathophysiology of cervical myelopathy. Spine J. 6, 190S–197S.

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