Journal of Cerebral Blood Flow & Metabolism (2015) 35, 977–984 © 2015 ISCBFM All rights reserved 0271-678X/15 $32.00 www.jcbfm.com
ORIGINAL ARTICLE
Lasting pure-motor deficits after focal posterior internal capsule white-matter infarcts in rats Francesco Blasi1, Michael J Whalen2,3 and Cenk Ayata1,4 Small white-matter infarcts of the internal capsule are clinically prevalent but underrepresented among currently available animal models of ischemic stroke. In particular, the assessment of long-term outcome, a primary end point in clinical practice, has been challenging due to mild deficits and the rapid and often complete recovery in most experimental models. We, therefore, sought to develop a focal white-matter infarction model that can mimic the lasting neurologic deficits commonly observed in stroke patients. The potent vasoconstrictor endothelin-1 (n = 24) or vehicle (n = 9) was stereotactically injected into the internal capsule at one of three antero-posterior levels (1, 2, or 3 mm posterior to bregma) in male Sprague-Dawley rats. Endothelin-injected animals showed highly focal (~1 mm3) and reproducible ischemic infarcts, with severe axonal and myelin loss accompanied by cellular infiltration when examined 2 and 4 weeks after injection. Only those rats injected with endothelin-1 at the most posterior location developed robust and pure-motor deficits in adhesive removal, cylinder and foot-fault tests that persisted at 1 month, without detectable sensory impairments. In summary, we present an internal capsule stroke model optimized to produce lasting pure-motor deficits in rats that may be suitable to study neurologic recovery and rehabilitation after white-matter injury. Journal of Cerebral Blood Flow & Metabolism (2015) 35, 977–984; doi:10.1038/jcbfm.2015.7; published online 4 February 2015 Keywords: endothelin-1; internal capsule; motor deficits; stroke; white matter
INTRODUCTION The severity of functional deficits after ischemic stroke strongly depends on infarct location.1 Isolated white-matter lesions involving the corticospinal tract are common among stroke patients, and lead to severe motor deficits, poor recovery, and unfavorable outcomes.2–4 This is especially the case when the posterior limb of internal capsule is involved where a large amount of dense pyramidal tract fibers project on to the spinal cord.5,6 Preclinical stroke research mostly relies on models of large territorial infarction affecting both white and gray matter. Isolated white-matter ischemia involving subcortical tracts has received relatively little attention and is underrepresented among the currently available models of ischemic stroke. This is in part due to the low white-matter content in rodent brain, as well as the technical challenges involved in producing a small strategically located lesion within a deep brain structure.7–9 In this context, a particularly useful approach is the stereotactic injection of endothelin-1 (ET-1), a potent vasoconstrictor that generates a focal ischemic lesion with both neuronal and glial degeneration.10 We recently showed that ET-1 injections within the peri-ventricular white matter (i.e., corpus callosum) lead to focal cerebral blood flow reduction and lasting tissue and cognitive deficits in mice.11 The ET-1 model has been used to produce capsular lesions in rats.12,13 However, deficits have been
mild and short-lasting, limiting the usefulness of such models for recovery paradigms. To address these shortcomings, we studied the tissue and functional outcome for up to 1 month after focal internal capsule ischemia produced by stereotactic ET-1 injection in rats, and examined the relationship between lesion localization and neurologic deficits. The data suggest that localization is critical to reproduce the lasting pure-motor deficits observed in patients with strokes involving the posterior limb of internal capsule.
MATERIALS AND METHODS Animals Experiments were conducted according to the ARRIVE guidelines,14 the protocols approved by the Animal Research Committee of Massachusetts General Hospital, and the National Institutes of Health ‘Guide for the Care and Use of Laboratory Animals’. Sprague-Dawley rats (males, 10 weeks old at the time of the surgery, Charles River Laboratories, Wilmington, MA, USA, n = 33) were housed two per cage and maintained on a standard light/dark cycle.
Internal Capsule Stroke Model Rats were anesthetized with isoflurane (4% for induction, 2.5% to 2% for maintenance, in 70%N2O/30%O2) and secured to a stereotactic frame (Stoelting, Wood Dale, IL, USA). Body temperature was maintained at 37°C
1 Neurovascular Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA; 2Neuroscience Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA; 3Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA and 4Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. Correspondence: Dr C Ayata, Neurovascular Research Laboratory, Massachusetts General Hospital, 149 13th Street, Room 6403, Charlestown, MA 02129, USA. E-mail: [email protected] This study was supported by the National Institutes of Health (NS061505 and NS055104), Fondation Leducq, Neuroendovascular Research Fund from the Andrew David Heitman Foundation, and The Ellison Foundation. Received 6 November 2014; revised 26 December 2014; accepted 30 December 2014; published online 4 February 2015
Motor deficit after capsular stroke in rats F Blasi et al
978 using a homeothermic blanket; eye ointment was applied to prevent corneal dryness. After a midline scalp incision, a small burr hole (1 × 1 mm) was drilled under constant saline cooling and 1 μL of ET-1 (0.3 mg/mL, American Peptide, Sunnyvale, CA, USA; stroke group, n = 24)11 or vehicle
(sterile saline; control group, n = 9) was injected using a 10-μL syringe (Hamilton, Reno, NV, USA; 33 gauge beveled needle) connected to a micropump (Stoelting; injection rate: 0.1 μL/min). The following stereotactic coordinates were targeted based on a rat brain atlas:15 ●
Table 1.
Time point Baseline 7 Days 14 Days 21 Days 28 Days
●
Body weights Stroke
Control
284 ± 4 305 ± 5 366 ± 9 431 ± 12 478 ± 14
287 ± 5 308 ± 4 365 ± 8 429 ± 8 464 ± 10
Values are in grams (mean ± s.e.m., n = 9 each). P40.05, two-way ANOVA for repeated measures followed by Bonferroni post hoc test.
●
Anterior (n = 7): AP − 1.0 mm; ML +5.5 mm; DV − 4.5 mm; angle of injection: 20°. Middle (n = 7): AP − 2.0 mm; ML +5.5 mm; DV − 5.0 mm; angle of injection: 18°. Posterior (n = 10): AP − 3.0 mm; ML +5.5 mm; DV − 5.0 mm; angle of injection: 15°.
An angled injection was preferred over a perpendicular approach to minimize the mechanical damage to primary motor, sensorimotor and hippocampal cortices, overlying the internal capsule.13 After the injection, the needle was left in place for additional 10 minutes to avoid backflow. The burr hole was sealed with bone wax (Ethicon, Somerville, NJ, USA). Rats were allowed to recover for 2 hours in a 28°C incubator before returning to the home cage.
Figure 1. Tissue outcome after endothelin-1 (ET-1) injection into the internal capsule. (A) Injection coordinates and angles at the anterior, middle, and posterior internal capsule (adapted from Paxinos and Watson).15 (B) Coronal sections stained with LFB-CV showing representative lesions after ET-1 injection at the anterior, middle, and posterior portions of the internal capsule. Infarct is well demarcated in the internal capsule (black dashed line) as well as in adjacent thalamus (red dashed line). (C) Total, internal capsule, and thalamic infarct areas in successive coronal sections are shown as a function of anteroposterior distance from bregma. Dotted lines represent injection sites. Error bars are s.e.m. (D) Box-and-whisker plots (whiskers, full range; box, 25% to 75%; line, median; cross, mean) showing total, internal capsule, and thalamic infarct volumes. Animals in anterior and middle groups were euthanized 14 days after stroke, while animals in posterior group were euthanized 28 days after stroke. Total infarct size was comparable among groups (P = 0.61). *P o0.05 versus posterior (one-way ANOVA followed by Bonferroni’s post hoc test). Scale bars: 2 mm. N = 7 anterior, 7 middle, and 9 posterior. CV, cresyl violet; IC, internal capsule; LFB, Luxol fast blue; Thal, thalamus; Tot, total. Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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Motor deficit after capsular stroke in rats F Blasi et al
979 Behavioral Assessments Blinded assessments were performed up to 4 weeks after ET-1 or vehicle injection. Rats were housed for 3 to 7 days in the animal facility before starting the behavioral assessments. In the same period, rats were handled to reduce the stress and exposed at least once to the experimental set-up. The arenas and the testing equipment were cleaned with 70% ethanol before each trial. All tests were performed in the active/exploratory phase, between 0800 and 1100 h or 1700 and 2000 h; rats were allowed to acclimate for 60 minutes in the behavioral room before starting the assessment. In the adhesive removal test,16 rats were gently held by the torso and a 1 × 2 cm piece of surgical tape (3 M Transpore, Auburn, MA, USA) was placed on the palmar surface of the forepaws. Time to contact and time to remove the tape were recorded from the mean of three trials per paw conducted by alternating the starting paw. An asymmetry index was obtained by the ratio between the time (seconds) spent to remove the tape from contralateral versus ipsilateral paws. In the cylinder test,16 rats were placed in a transparent plastic cylinder (diameter: 20 cm), which the animal rears to explore. The number of wall touches with right, left, and both forepaws was recorded. Only rears in which both paws could be clearly seen were counted. The percentage of ipsilateral or contralateral contacts over the total number of wall contacts was then calculated. An asymmetry index was obtained by the ratio between the percentages of ipsilateral versus contralateral contacts. For the foot-fault test,17 each rat was placed atop an elevated wire grid (3 × 3 cm wire mesh) and video-recorded while freely walking on the grid for 10 minutes. A total of 100 to 150 steps were counted. The number of foot-faults was recorded and expressed as percent of total steps. For the corner test,17 animals were placed facing a corner with 30° angle formed by two walls and with a small opening at the joint. As the animal approaches the corner, vibrissae are stimulated that leads the animal to rear and turn around. Left (ipsilateral) and right (contralateral) turns were counted over 10 trials. For the open field test,18 rats were allowed to freely explore an arena (60 × 60 cm) for 5 minutes; exploratory activity was analyzed using the ANY-MAZE tracking system (Stoelting). Finally, sensory deficits were examined using the Hargreaves’ test.19 A radiant heat beam (stimulus cutoff: 20 seconds; intensity: IR30) was applied to the mid-plantar surface of each hindpaw from underneath a glass floor with a projector lamp bulb (Stoelting). Two trials were performed on each paw, by alternating the starting paw, with an interval of at least 5 minutes. A positive pain reaction was defined as sudden paw withdrawal, flinching, and paw licking induced by the heat application. Latencies to withdraw were averaged between the two trials for each paw.
Histopathology and Infarct Size Assessment At 14 or 28 days after stroke rats were euthanized, brains harvested and snap-frozen in isopentane (−45°C), cryosectioned (section interval 200 μm) and fixed in absolute alcohol for 10 minutes at room temperature. Luxol fast blue (myelin) staining was performed by incubating the sections in an alcoholic solution of Solvent Blue 38 (0.1% w/v, Sigma Aldrich, St Louis, MO, USA, cat. S3382) at 60°C for 2 hours. Differentiation was obtained
using an aqueous solution of lithium carbonate (0.05% w/v, Poly Scientific, Bay Shore, NY, USA, S2048). Sections were counterstained with Cresyl Violet acetate (nuclear marker, 0.5% w/v, Sigma Aldrich, C5042). Hematoxylin and eosin (H&E, general neuropathology) staining was performed according to standard protocols. Mouse monoclonal antimyelin basic protein (MSB, oligodendrocytes, 4 μg/mL, Covance, Dedham, MA, USA, SMI-94R) and rabbit polyclonal anti-neurofilament 200 (NF200, axons, 16 μg/mL, Sigma Aldrich, N4142) antibodies were co-incubated overnight at 4°C, in 3% horse serum, PBS 0.01 mol/L, pH 7.4. Donkey antimouse secondary antibody-Alexa488 conjugated (10 μg/mL, Molecular Probes, Eugene, OR, USA, A-21202) and goat anti-rabbit secondary antibody-Cy3 conjugated (7.5 μg/mL, Jackson ImmunoResearch, West Grove, PA, USA, 111-165-003) were co-incubated for 60 minutes at room temperature, in PBS 0.01 mol/L, pH 7.4. Hoechst 33342 (nuclei, 1 μg/mL, Sigma Aldrich, 14533) was incubated for 10 minutes at room temperature, in PBS 0.01 mol/L, pH 7.4. Images were acquired using a Super Coolscan 9000 ED Scanner (Nikon, Melville, NY, USA) and a TE-2000 microscope equipped with epifluorescence illumination (Nikon). Staining conditions and acquisition parameters were comparable for all samples. Capsular infarct was quantified by using ImageJ (NIH, Bethesda, MD, USA). The infarct area was measured at each section level (every 200 μm) and integrated across the lesion to calculate the volume.11 Brain edema was not detected at the time points investigated.
Experimental Design After surgery, rats were coded with tail marks by an investigator blinded to the group identity; blinding was maintained until the end of data collection and analysis. Sample size was calculated to detect a group difference of 40% in neurologic deficits, power 90%, alpha 0.05 and a standard deviation of 40% of the mean, based on pilot data assuming normal distribution. Randomization was not performed since no drug testing was performed. A priori exclusion criteria were poor body condition and weight loss greater than 30%, seizures, and technical failure during injection. Body weights were comparable between stroke and control groups at every time point (Table 1). Arterial blood pressure, pH, and blood gases were not monitored to avoid behavioral confounders due to the arterial catheterization; intracerebral ET-1 has not altered systemic physiologic parameters in previous studies.10 Experiments were performed in three steps. In initial pilot experiments, three rats were injected with ET-1 at each of the three internal capsule locations to determine the optimal site of injection to produce consistent motor deficits (step 1); adhesive removal, cylinder, foot-fault, and corner tests were performed in this initial group at baseline and 7 and 14 days after the injection. After identifying the posterior injection site as the preferred location, observation period for the three rats in the posterior injection group was extended to 28 days, and seven more rats were added along with nine vehicle controls (step 2); therefore, the complete battery of neurologic tests were studied in the posterior injection group. In step 3, four more rats were added to the anterior and middle injection groups, in which only
Figure 2. Internal capsule injection site and motor deficits. (A) Adhesive removal test shows the asymmetry index expressed as a ratio of the time spent removing the tape from contralateral versus ipsilateral forepaw. (B) Cylinder test shows the forelimb asymmetry (ratio of ipsilateral versus contralateral contacts) during wall exploration. N = 7 anterior (ANT), 7 middle, and 9 posterior (POST). Dashed lines indicate normal function (i.e., no asymmetry). *P o0.05 versus anterior and middle, #Po 0.05 versus baseline, two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Error bars are s.e.m.
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Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
Motor deficit after capsular stroke in rats F Blasi et al
980 adhesive removal and cylinder tests were studied for up to 14 days. There was no spontaneous mortality associated with this model assessed up to 28 days after stroke. Only one animal was excluded due to seizures, weight loss, and poor body condition (posterior injection), possibly caused by ET-1 diffusion into the lateral ventricle.20 Therefore, altogether, a total of seven rats injected with ET-1 were studied for 14 days in the anterior and middle injection groups, and nine rats injected with ET-1 or vehicle each were studied for 28 days in the posterior injection group.
Statistics Data were normally distributed (D’Agostino and Pearson omnibus test for normality, P40.05), and expressed as mean ± s.e.m. or whisker box
plots. Differences between groups were compared using one-way ANOVA or two-way ANOVA for repeated measures followed by Bonferroni’s post hoc test, where appropriate. Student’s t-test was used to assess open field and Hargreaves tests. Po0.05 was considered as significant.
RESULTS Infarct Location To determine the infarct location(s) in the internal capsule that yielded reproducible motor deficits after stroke, we injected ET-1 into the left anterior, middle or posterior internal capsule in three
Figure 3. Long-term motor deficits after posterior internal capsule stroke. (A) Contact and removal times assessed with adhesive removal test. (B) Forelimb asymmetry during wall exploration assessed with cylinder test. (C) Foot-faults (%) over total steps taken during grid exploration. (D) Turning asymmetry in corner test. (E) Spontaneous exploration assessed using open field test. (F) Contralateral thermal hypersensitivity assessed with Hargreaves test. N = 9 each. Dashed line shows normal function (i.e., no asymmetry). For box-and-whisker plots: whiskers, full range; box, 25% to 75%; line, median; cross, mean. *Po0.05 versus control, #Po 0.05 versus ipsilateral to the lesion in stroke group, two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Error bars are s.e.m. Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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Motor deficit after capsular stroke in rats F Blasi et al
981 different groups. Histologic examination showed that all lesions were highly focal (~1 mm3), and involved and interrupted the internal capsule in all groups (Figure 1B; black dashed line). Although the total infarct size was comparable among the groups (Figure 1C), posterior lesions involved larger volumes of the internal capsule, and anterior and middle lesions showed larger involvement of the lateral thalamic nuclei adjacent to the internal capsule (Figure 1B; red dashed line). ET-1 lesions never involved cortex, striatum, or substantia nigra. Notably, all lesions extended posteriorly from the injection coordinates (Figure 1C), possibly due to a tendency for ET-1 to diffuse posteriorly after injection or an anterior to posterior flow direction of end arteries supplying the internal capsule. Neurologic Deficits Baseline neurologic assessments did not reveal any difference among the groups and between ipsilateral and contralateral paws. Endothelin-1 injection into the posterior internal capsule (bregma − 3 mm) consistently induced a robust asymmetry in forelimb use in both adhesive removal and cylinder tests compared with baseline, which persisted for 14 days (Figure 2). In contrast, anterior (bregma − 1 mm) and middle (bregma − 2 mm) internal capsule ET-1 injections did not cause any deficits detectable using these tests at any time point during the 14-day follow-up. Therefore, we focused the rest of the study on the posterior group only and extended the follow-up period to 28 days. Neurologic deficits after posterior internal capsule lesion were most severe when first assessed 7 days after stroke and persisted for at least 28 days (Figure 3). Deficits were pure motor, detected using the
adhesive removal, cylinder, and foot-fault tests. At 7 days after injection, the contralateral removal time was three times greater than the ipsilateral removal time, as well as the contralateral removal time in vehicle controls, and remained significantly elevated at 28 days. The cylinder test showed 5- and 3-fold greater forelimb asymmetry at 7 and 28 days, respectively. The percentage of foot-faults during grid exploration was greater in stroke animals than controls at both early and late time points. Importantly, the percentage of foot-faults decreased gradually in control animals as a reflection of task habituation and learning; this improvement was absent in the stroke group. Sensory function was not impaired, with normal contact times in adhesive removal test, normal turning preference in corner test, and normal contralateral thermal sensitivity in Hargreaves test. Spontaneous exploratory activity in open field test also did not differ between the groups, ruling out behavioral confounders linked to reduced locomotion. Histopathology Four weeks after ET-1 injection, the internal capsule infarct showed abundant white-matter loss (Figure 4A). The H&E staining revealed a cellular infiltrate (asterisk) at the infarct site (Figures 4B and 4C). White-matter injury was not seen in vehicle-injected control rats (Figures 4D to 4F). Immunofluorescence revealed severe loss of myelin (MBP) and axons (NF200) within the infarct (Figures 5A and 5B); some NF200 immunoreactivity was still present in the infarct possibly due to residual antigen from degenerated fibers.21 The areas of myelin and axonal loss were congruent (Figure 5C), and there was an abrupt demarcation
Figure 4. Tissue outcome 1 month after posterior internal capsule stroke. (A) White-matter infarct (arrow) 28 days after endothelin-1 injection revealed by LFB-CV staining. (B) Hematoxylin and eosin (H&E)-stained coronal section showing internal capsule lesion and high cellularity (asterisk) at the level of the infarct site. (C) High magnification image from the region marked with the asterisk reveals widespread cellular infiltration. (D–F) Absence of white-matter damage and cellular infiltration after vehicle injection. Histology was similar for all investigated animals (n = 9/group). Scale bars: 1 mm (A, B, D, and E), 0.1 mm (C and F). LFB, Luxol fast blue. © 2015 ISCBFM
Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
Motor deficit after capsular stroke in rats F Blasi et al
982 between the infarct and surrounding intact tissue at this late time point (Figure 5D). However, axonal density appeared to be reduced in all animals even in the peri-infarct tissue. This may be due to Wallerian degeneration slowly developing over days to weeks, or there may be slow, delayed, and selective axonal loss in the peri-infarct region where ischemia was milder (i.e., penumbra). Vehicle injection did not result in myelin and axonal changes in the internal capsule (Figures 5E to 5H).
DISCUSSION Here, we show that a highly focal white matter infarct in the posterior limb of internal capsule leads to pronounced and puremotor deficits that persist for at least 1 month in rats. The infarct location, lasting motor deficits, and lack of detectable sensory impairments observed in the present model resemble the typical human pure-motor syndrome associated with lacunar infarcts of the internal capsule. Therefore, stereotactic ET-1 injection is a
Figure 5. Myelin and axonal loss in the internal capsule after stroke. Immunofluorescence stainings for (A) myelin basic protein (MBP) and (B) neurofilament-200 (NF200) reveal myelin and axonal degeneration in the internal capsule (arrows) 1 month after stroke. (C) Triple fluorescence image showing congruent loss of MBP and NF200 immunoreactive fibers in the infarct core (arrow), and high cellularity detected with Hoechst 33342 (nuclei, blue). (D) High magnification image from the region marked with the asterisk in (B) showing axonal morphology in the periinfarct internal capsule and degenerated fibers in the infarct. (E–H) Normal white-matter morphology in the internal capsule after vehicle injection. Histology was similar for all investigated animals (n = 9/group). Scale bars: 0.5 mm (A–C and E–G), 0.05 mm (D and H). Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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Motor deficit after capsular stroke in rats F Blasi et al
983 suitable model to investigate the long-term functional outcome after focal ischemic white-matter capsular lesions. Although a significant portion of ischemic strokes involves the white matter, experimental modeling of focal white-matter injury has lagged behind large artery occlusion models. This has been in part because selective targeting of small volumes of white matter in rodent brain is technically challenging. Only about 15% to 20% of rodent brain volume is white matter, compared with 50% in human brain.8 Moreover, vascular anatomy is not permissive to selectively interrupt blood supply to white matter in rodents. Internal capsule is perfused by the anterior choroidal artery and the lenticulostriate branches of the middle cerebral artery in both human and rodents.22 Although selective occlusion of the anterior choroidal artery in pigs has created small infarcts mainly involving the internal capsule,23 in rodents infarcts have also involved thalamus and hypothalamus, thereby limiting the usefulness of this approach.24 Global hypoperfusion (e.g., bilateral carotid occlusion in rats25 or stenosis in mice26) does lead to diffuse blood flow reduction, white-matter rarefaction, and cumulative ischemic damage, but lesions mimic more chronic progressive leukoaraiosis rather than sudden-onset focal ischemic whitematter infarction. Photothrombosis has recently been used to target the internal capsule causing motor deficits lasting at least 3 weeks.27 However, sensory function was not examined in this study, and lesions also involved the entopeduncular nucleus (corresponding to the human globus pallidus internus) that may lead to motor deficits and were adjacent to the optic tract that may cause vision defects. Moreover, mechanisms of photothrombotic injury may involve more than just tissue ischemia. Finally, selective white-matter lesions can also be produced by focal injection of toxins targeting oligodendrocytes (e.g., lysolecithin injections28), but these lesions are predominantly demyelinating rather than ischemic. Compared with above, stereotactic injection of ET-1 (or other potent vasoconstrictors)29 is a unique and superior method to generate highly focal ischemia that can be strategically located to produce white-matter infarcts.10–13,30 However, lasting pure-motor deficits after a strategically located lesion have never been demonstrated. In one study,12 motor deficits were limited to a modest twofold increase in foot-faults, and sensory deficits were more prominent, possibly due to the lesion placement in anterior dorsal internal capsule. In another study in hypertensive rats,13 lesions were smaller, and motor deficits were once again very mild (cylinder test asymmetry index ~ 1.5) and limited to the forelimb (walking ladder test score ~ 5.3 on a scale of 0 worst to 6 best). Both studies followed the animals for only 2 weeks or less; therefore, persistence of the deficits was not demonstrated. Therefore, our model offers distinct and novel advantages over previously published ones. First, motor deficits were significantly more severe (e.g., threefold increase in foot-faults, cylinder test asymmetry index ~ 4), likely reflecting the optimized lesion location in the posterior internal capsule as well as larger infarct sizes (~1 mm3). That posterior lesions caused greater motor deficits in our study is in line with worse motor outcomes after strokes involving the posterior limb of internal capsule, presumably due to interruption of the dense corticospinal tract projections.3,4 Second, we showed that the deficits persisted for at least 28 days. Importantly, in the posterior injection group more than 90% of the infarct was within the internal capsule, and the small thalamic involvement did not cause sensory deficits in forelimb tactile sensitivity (contact time in adhesive removal test), vibrissae symmetry (corner test), and hind limb thermal sensitivity (Hargreaves test). The absence of mortality and good reproducibility (coefficient of variation ~ 30% for infarct volume, ~ 40% for neurologic deficits) make the model especially suitable to study older and comorbid animals,31 which are critical for clinical translation but expensive and difficult to procure. Finally, the absence of injury in other motor areas of the brain (e.g., cortex, © 2015 ISCBFM
striatum, and substantia nigra), as well as areas that may confound behavioral tests (e.g., optic nerve and tract, hippocampus), is an advantage over other models that result in white-matter injury, such as chronic forebrain hypoperfusion, chronic hypertension, intracarotid sodium laurate injection, or capsular photothrombotic lesion.9,27,32–36 Of course, ET-1 model has limitations. The mechanism of ischemia does not mimic lacunar pathophysiology, multiple vessels constrict at the same time, the duration of ischemia cannot be precisely controlled, and ET-1 targets not only endothelial and smooth muscle cells but also neurons and glia as a possible confounder.10,37 Lacunar infarcts account for 25% of all ischemic strokes,38 and are caused by occlusion of a single penetrating artery due to microatheroma or lipohyalinosis,39 although this is still debated.40 None of the existing whitematter stroke models accurately recapitulates small vessel disease and lacunar stroke mechanisms.32 For these reasons, the model is better suited to investigate recovery in chronic stages, as well as ischemic white-matter injury progression and repair after the hyperacute stage (e.g., 412 hours), which differs from gray matter given the predominantly oligodendrocyte and axon injury rather than neuronal cell bodies.9 Functional outcome is a primary end point after stroke, but repair and recovery after white-matter stroke are still poorly understood due to a lack of experimental models that yield reproducible and lasting functional deficits. Therefore, our model represents a critical improvement to facilitate preclinical investigations of novel therapeutic interventions targeting recovery after white-matter injury. DISCLOSURE/CONFLICT OF INTEREST The authors declare no conflict of interest.
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research: reporting in vivo experiments--the ARRIVE guidelines. J Cereb Blood Flow Metab 2011; 31: 991–993. Paxinos G, Watson C. The rat brain in stereotaxic coordinates 6th edn. Elsevier: San Diego, 2008. Schallert T. Behavioral tests for preclinical intervention assessment. NeuroRx 2006; 3: 497–504. Schaar KL, Brenneman MM, Savitz SI. Functional assessments in the rodent stroke model. Exp Transl Stroke Med 2010; 2: 13. Walsh RN, Cummins RA. The Open-Field Test: a critical review. Psychol Bull 1976; 83: 482–504. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988; 32: 77–88. Chew BH, Weaver DF, Gross PM. Dose-related potent brain stimulation by the neuropeptide endothelin-1 after intraventricular administration in conscious rats. Pharmacol Biochem Behav 1995; 51: 37–47. Girard C, Liu S, Cadepond F, Adams D, Lacroix C, Verleye M et al. Etifoxine improves peripheral nerve regeneration and functional recovery. Proc Natl Acad Sci USA 2008; 105: 20505–20510. He Z, Yang S-H, Naritomi H, Yamawaki T, Liu Q, King MA et al. Definition of the anterior choroidal artery territory in rats using intraluminal occluding technique. J Neurol Sci 2000; 182: 16–28. Tanaka Y, Imai H, Konno K, Miyagishima T, Kubota C, Puentes S et al. Experimental model of lacunar infarction in the gyrencephalic brain of the miniature pig: neurological assessment and histological, immunohistochemical, and physiological evaluation of dynamic corticospinal tract deformation. Stroke 2008; 39: 205–212. He Z, Yamawaki T, Yang S, Day AL, Simpkins JW, Naritomi H et al.. Experimental model of small deep infarcts involving the hypothalamus in rats: changes in body temperature and postural reflex. Stroke 1999; 30: 2743–2751. Wakita H, Tomimoto H, Akiguchi I, Kimura J. Glial activation and white matter changes in the rat brain induced by chronic cerebral hypoperfusion: an immunohistochemical study. Acta Neuropathol 1994; 87: 484–492. Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke 2004; 35: 2598–2603. Kim HS, Kim D, Kim RG, Kim JM, Chung E, Neto PR et al. A rat model of photothrombotic capsular infarct with a marked motor deficit: a behavioral,
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ORIGINAL ARTICLE
Lasting pure-motor deficits after focal posterior internal capsule white-matter infarcts in rats Francesco Blasi1, Michael J Whalen2,3 and Cenk Ayata1,4 Small white-matter infarcts of the internal capsule are clinically prevalent but underrepresented among currently available animal models of ischemic stroke. In particular, the assessment of long-term outcome, a primary end point in clinical practice, has been challenging due to mild deficits and the rapid and often complete recovery in most experimental models. We, therefore, sought to develop a focal white-matter infarction model that can mimic the lasting neurologic deficits commonly observed in stroke patients. The potent vasoconstrictor endothelin-1 (n = 24) or vehicle (n = 9) was stereotactically injected into the internal capsule at one of three antero-posterior levels (1, 2, or 3 mm posterior to bregma) in male Sprague-Dawley rats. Endothelin-injected animals showed highly focal (~1 mm3) and reproducible ischemic infarcts, with severe axonal and myelin loss accompanied by cellular infiltration when examined 2 and 4 weeks after injection. Only those rats injected with endothelin-1 at the most posterior location developed robust and pure-motor deficits in adhesive removal, cylinder and foot-fault tests that persisted at 1 month, without detectable sensory impairments. In summary, we present an internal capsule stroke model optimized to produce lasting pure-motor deficits in rats that may be suitable to study neurologic recovery and rehabilitation after white-matter injury. Journal of Cerebral Blood Flow & Metabolism (2015) 35, 977–984; doi:10.1038/jcbfm.2015.7; published online 4 February 2015 Keywords: endothelin-1; internal capsule; motor deficits; stroke; white matter
INTRODUCTION The severity of functional deficits after ischemic stroke strongly depends on infarct location.1 Isolated white-matter lesions involving the corticospinal tract are common among stroke patients, and lead to severe motor deficits, poor recovery, and unfavorable outcomes.2–4 This is especially the case when the posterior limb of internal capsule is involved where a large amount of dense pyramidal tract fibers project on to the spinal cord.5,6 Preclinical stroke research mostly relies on models of large territorial infarction affecting both white and gray matter. Isolated white-matter ischemia involving subcortical tracts has received relatively little attention and is underrepresented among the currently available models of ischemic stroke. This is in part due to the low white-matter content in rodent brain, as well as the technical challenges involved in producing a small strategically located lesion within a deep brain structure.7–9 In this context, a particularly useful approach is the stereotactic injection of endothelin-1 (ET-1), a potent vasoconstrictor that generates a focal ischemic lesion with both neuronal and glial degeneration.10 We recently showed that ET-1 injections within the peri-ventricular white matter (i.e., corpus callosum) lead to focal cerebral blood flow reduction and lasting tissue and cognitive deficits in mice.11 The ET-1 model has been used to produce capsular lesions in rats.12,13 However, deficits have been
mild and short-lasting, limiting the usefulness of such models for recovery paradigms. To address these shortcomings, we studied the tissue and functional outcome for up to 1 month after focal internal capsule ischemia produced by stereotactic ET-1 injection in rats, and examined the relationship between lesion localization and neurologic deficits. The data suggest that localization is critical to reproduce the lasting pure-motor deficits observed in patients with strokes involving the posterior limb of internal capsule.
MATERIALS AND METHODS Animals Experiments were conducted according to the ARRIVE guidelines,14 the protocols approved by the Animal Research Committee of Massachusetts General Hospital, and the National Institutes of Health ‘Guide for the Care and Use of Laboratory Animals’. Sprague-Dawley rats (males, 10 weeks old at the time of the surgery, Charles River Laboratories, Wilmington, MA, USA, n = 33) were housed two per cage and maintained on a standard light/dark cycle.
Internal Capsule Stroke Model Rats were anesthetized with isoflurane (4% for induction, 2.5% to 2% for maintenance, in 70%N2O/30%O2) and secured to a stereotactic frame (Stoelting, Wood Dale, IL, USA). Body temperature was maintained at 37°C
1 Neurovascular Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA; 2Neuroscience Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA; 3Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA and 4Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. Correspondence: Dr C Ayata, Neurovascular Research Laboratory, Massachusetts General Hospital, 149 13th Street, Room 6403, Charlestown, MA 02129, USA. E-mail: [email protected] This study was supported by the National Institutes of Health (NS061505 and NS055104), Fondation Leducq, Neuroendovascular Research Fund from the Andrew David Heitman Foundation, and The Ellison Foundation. Received 6 November 2014; revised 26 December 2014; accepted 30 December 2014; published online 4 February 2015
Motor deficit after capsular stroke in rats F Blasi et al
978 using a homeothermic blanket; eye ointment was applied to prevent corneal dryness. After a midline scalp incision, a small burr hole (1 × 1 mm) was drilled under constant saline cooling and 1 μL of ET-1 (0.3 mg/mL, American Peptide, Sunnyvale, CA, USA; stroke group, n = 24)11 or vehicle
(sterile saline; control group, n = 9) was injected using a 10-μL syringe (Hamilton, Reno, NV, USA; 33 gauge beveled needle) connected to a micropump (Stoelting; injection rate: 0.1 μL/min). The following stereotactic coordinates were targeted based on a rat brain atlas:15 ●
Table 1.
Time point Baseline 7 Days 14 Days 21 Days 28 Days
●
Body weights Stroke
Control
284 ± 4 305 ± 5 366 ± 9 431 ± 12 478 ± 14
287 ± 5 308 ± 4 365 ± 8 429 ± 8 464 ± 10
Values are in grams (mean ± s.e.m., n = 9 each). P40.05, two-way ANOVA for repeated measures followed by Bonferroni post hoc test.
●
Anterior (n = 7): AP − 1.0 mm; ML +5.5 mm; DV − 4.5 mm; angle of injection: 20°. Middle (n = 7): AP − 2.0 mm; ML +5.5 mm; DV − 5.0 mm; angle of injection: 18°. Posterior (n = 10): AP − 3.0 mm; ML +5.5 mm; DV − 5.0 mm; angle of injection: 15°.
An angled injection was preferred over a perpendicular approach to minimize the mechanical damage to primary motor, sensorimotor and hippocampal cortices, overlying the internal capsule.13 After the injection, the needle was left in place for additional 10 minutes to avoid backflow. The burr hole was sealed with bone wax (Ethicon, Somerville, NJ, USA). Rats were allowed to recover for 2 hours in a 28°C incubator before returning to the home cage.
Figure 1. Tissue outcome after endothelin-1 (ET-1) injection into the internal capsule. (A) Injection coordinates and angles at the anterior, middle, and posterior internal capsule (adapted from Paxinos and Watson).15 (B) Coronal sections stained with LFB-CV showing representative lesions after ET-1 injection at the anterior, middle, and posterior portions of the internal capsule. Infarct is well demarcated in the internal capsule (black dashed line) as well as in adjacent thalamus (red dashed line). (C) Total, internal capsule, and thalamic infarct areas in successive coronal sections are shown as a function of anteroposterior distance from bregma. Dotted lines represent injection sites. Error bars are s.e.m. (D) Box-and-whisker plots (whiskers, full range; box, 25% to 75%; line, median; cross, mean) showing total, internal capsule, and thalamic infarct volumes. Animals in anterior and middle groups were euthanized 14 days after stroke, while animals in posterior group were euthanized 28 days after stroke. Total infarct size was comparable among groups (P = 0.61). *P o0.05 versus posterior (one-way ANOVA followed by Bonferroni’s post hoc test). Scale bars: 2 mm. N = 7 anterior, 7 middle, and 9 posterior. CV, cresyl violet; IC, internal capsule; LFB, Luxol fast blue; Thal, thalamus; Tot, total. Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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979 Behavioral Assessments Blinded assessments were performed up to 4 weeks after ET-1 or vehicle injection. Rats were housed for 3 to 7 days in the animal facility before starting the behavioral assessments. In the same period, rats were handled to reduce the stress and exposed at least once to the experimental set-up. The arenas and the testing equipment were cleaned with 70% ethanol before each trial. All tests were performed in the active/exploratory phase, between 0800 and 1100 h or 1700 and 2000 h; rats were allowed to acclimate for 60 minutes in the behavioral room before starting the assessment. In the adhesive removal test,16 rats were gently held by the torso and a 1 × 2 cm piece of surgical tape (3 M Transpore, Auburn, MA, USA) was placed on the palmar surface of the forepaws. Time to contact and time to remove the tape were recorded from the mean of three trials per paw conducted by alternating the starting paw. An asymmetry index was obtained by the ratio between the time (seconds) spent to remove the tape from contralateral versus ipsilateral paws. In the cylinder test,16 rats were placed in a transparent plastic cylinder (diameter: 20 cm), which the animal rears to explore. The number of wall touches with right, left, and both forepaws was recorded. Only rears in which both paws could be clearly seen were counted. The percentage of ipsilateral or contralateral contacts over the total number of wall contacts was then calculated. An asymmetry index was obtained by the ratio between the percentages of ipsilateral versus contralateral contacts. For the foot-fault test,17 each rat was placed atop an elevated wire grid (3 × 3 cm wire mesh) and video-recorded while freely walking on the grid for 10 minutes. A total of 100 to 150 steps were counted. The number of foot-faults was recorded and expressed as percent of total steps. For the corner test,17 animals were placed facing a corner with 30° angle formed by two walls and with a small opening at the joint. As the animal approaches the corner, vibrissae are stimulated that leads the animal to rear and turn around. Left (ipsilateral) and right (contralateral) turns were counted over 10 trials. For the open field test,18 rats were allowed to freely explore an arena (60 × 60 cm) for 5 minutes; exploratory activity was analyzed using the ANY-MAZE tracking system (Stoelting). Finally, sensory deficits were examined using the Hargreaves’ test.19 A radiant heat beam (stimulus cutoff: 20 seconds; intensity: IR30) was applied to the mid-plantar surface of each hindpaw from underneath a glass floor with a projector lamp bulb (Stoelting). Two trials were performed on each paw, by alternating the starting paw, with an interval of at least 5 minutes. A positive pain reaction was defined as sudden paw withdrawal, flinching, and paw licking induced by the heat application. Latencies to withdraw were averaged between the two trials for each paw.
Histopathology and Infarct Size Assessment At 14 or 28 days after stroke rats were euthanized, brains harvested and snap-frozen in isopentane (−45°C), cryosectioned (section interval 200 μm) and fixed in absolute alcohol for 10 minutes at room temperature. Luxol fast blue (myelin) staining was performed by incubating the sections in an alcoholic solution of Solvent Blue 38 (0.1% w/v, Sigma Aldrich, St Louis, MO, USA, cat. S3382) at 60°C for 2 hours. Differentiation was obtained
using an aqueous solution of lithium carbonate (0.05% w/v, Poly Scientific, Bay Shore, NY, USA, S2048). Sections were counterstained with Cresyl Violet acetate (nuclear marker, 0.5% w/v, Sigma Aldrich, C5042). Hematoxylin and eosin (H&E, general neuropathology) staining was performed according to standard protocols. Mouse monoclonal antimyelin basic protein (MSB, oligodendrocytes, 4 μg/mL, Covance, Dedham, MA, USA, SMI-94R) and rabbit polyclonal anti-neurofilament 200 (NF200, axons, 16 μg/mL, Sigma Aldrich, N4142) antibodies were co-incubated overnight at 4°C, in 3% horse serum, PBS 0.01 mol/L, pH 7.4. Donkey antimouse secondary antibody-Alexa488 conjugated (10 μg/mL, Molecular Probes, Eugene, OR, USA, A-21202) and goat anti-rabbit secondary antibody-Cy3 conjugated (7.5 μg/mL, Jackson ImmunoResearch, West Grove, PA, USA, 111-165-003) were co-incubated for 60 minutes at room temperature, in PBS 0.01 mol/L, pH 7.4. Hoechst 33342 (nuclei, 1 μg/mL, Sigma Aldrich, 14533) was incubated for 10 minutes at room temperature, in PBS 0.01 mol/L, pH 7.4. Images were acquired using a Super Coolscan 9000 ED Scanner (Nikon, Melville, NY, USA) and a TE-2000 microscope equipped with epifluorescence illumination (Nikon). Staining conditions and acquisition parameters were comparable for all samples. Capsular infarct was quantified by using ImageJ (NIH, Bethesda, MD, USA). The infarct area was measured at each section level (every 200 μm) and integrated across the lesion to calculate the volume.11 Brain edema was not detected at the time points investigated.
Experimental Design After surgery, rats were coded with tail marks by an investigator blinded to the group identity; blinding was maintained until the end of data collection and analysis. Sample size was calculated to detect a group difference of 40% in neurologic deficits, power 90%, alpha 0.05 and a standard deviation of 40% of the mean, based on pilot data assuming normal distribution. Randomization was not performed since no drug testing was performed. A priori exclusion criteria were poor body condition and weight loss greater than 30%, seizures, and technical failure during injection. Body weights were comparable between stroke and control groups at every time point (Table 1). Arterial blood pressure, pH, and blood gases were not monitored to avoid behavioral confounders due to the arterial catheterization; intracerebral ET-1 has not altered systemic physiologic parameters in previous studies.10 Experiments were performed in three steps. In initial pilot experiments, three rats were injected with ET-1 at each of the three internal capsule locations to determine the optimal site of injection to produce consistent motor deficits (step 1); adhesive removal, cylinder, foot-fault, and corner tests were performed in this initial group at baseline and 7 and 14 days after the injection. After identifying the posterior injection site as the preferred location, observation period for the three rats in the posterior injection group was extended to 28 days, and seven more rats were added along with nine vehicle controls (step 2); therefore, the complete battery of neurologic tests were studied in the posterior injection group. In step 3, four more rats were added to the anterior and middle injection groups, in which only
Figure 2. Internal capsule injection site and motor deficits. (A) Adhesive removal test shows the asymmetry index expressed as a ratio of the time spent removing the tape from contralateral versus ipsilateral forepaw. (B) Cylinder test shows the forelimb asymmetry (ratio of ipsilateral versus contralateral contacts) during wall exploration. N = 7 anterior (ANT), 7 middle, and 9 posterior (POST). Dashed lines indicate normal function (i.e., no asymmetry). *P o0.05 versus anterior and middle, #Po 0.05 versus baseline, two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Error bars are s.e.m.
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Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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980 adhesive removal and cylinder tests were studied for up to 14 days. There was no spontaneous mortality associated with this model assessed up to 28 days after stroke. Only one animal was excluded due to seizures, weight loss, and poor body condition (posterior injection), possibly caused by ET-1 diffusion into the lateral ventricle.20 Therefore, altogether, a total of seven rats injected with ET-1 were studied for 14 days in the anterior and middle injection groups, and nine rats injected with ET-1 or vehicle each were studied for 28 days in the posterior injection group.
Statistics Data were normally distributed (D’Agostino and Pearson omnibus test for normality, P40.05), and expressed as mean ± s.e.m. or whisker box
plots. Differences between groups were compared using one-way ANOVA or two-way ANOVA for repeated measures followed by Bonferroni’s post hoc test, where appropriate. Student’s t-test was used to assess open field and Hargreaves tests. Po0.05 was considered as significant.
RESULTS Infarct Location To determine the infarct location(s) in the internal capsule that yielded reproducible motor deficits after stroke, we injected ET-1 into the left anterior, middle or posterior internal capsule in three
Figure 3. Long-term motor deficits after posterior internal capsule stroke. (A) Contact and removal times assessed with adhesive removal test. (B) Forelimb asymmetry during wall exploration assessed with cylinder test. (C) Foot-faults (%) over total steps taken during grid exploration. (D) Turning asymmetry in corner test. (E) Spontaneous exploration assessed using open field test. (F) Contralateral thermal hypersensitivity assessed with Hargreaves test. N = 9 each. Dashed line shows normal function (i.e., no asymmetry). For box-and-whisker plots: whiskers, full range; box, 25% to 75%; line, median; cross, mean. *Po0.05 versus control, #Po 0.05 versus ipsilateral to the lesion in stroke group, two-way ANOVA for repeated measures followed by Bonferroni post hoc test. Error bars are s.e.m. Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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Motor deficit after capsular stroke in rats F Blasi et al
981 different groups. Histologic examination showed that all lesions were highly focal (~1 mm3), and involved and interrupted the internal capsule in all groups (Figure 1B; black dashed line). Although the total infarct size was comparable among the groups (Figure 1C), posterior lesions involved larger volumes of the internal capsule, and anterior and middle lesions showed larger involvement of the lateral thalamic nuclei adjacent to the internal capsule (Figure 1B; red dashed line). ET-1 lesions never involved cortex, striatum, or substantia nigra. Notably, all lesions extended posteriorly from the injection coordinates (Figure 1C), possibly due to a tendency for ET-1 to diffuse posteriorly after injection or an anterior to posterior flow direction of end arteries supplying the internal capsule. Neurologic Deficits Baseline neurologic assessments did not reveal any difference among the groups and between ipsilateral and contralateral paws. Endothelin-1 injection into the posterior internal capsule (bregma − 3 mm) consistently induced a robust asymmetry in forelimb use in both adhesive removal and cylinder tests compared with baseline, which persisted for 14 days (Figure 2). In contrast, anterior (bregma − 1 mm) and middle (bregma − 2 mm) internal capsule ET-1 injections did not cause any deficits detectable using these tests at any time point during the 14-day follow-up. Therefore, we focused the rest of the study on the posterior group only and extended the follow-up period to 28 days. Neurologic deficits after posterior internal capsule lesion were most severe when first assessed 7 days after stroke and persisted for at least 28 days (Figure 3). Deficits were pure motor, detected using the
adhesive removal, cylinder, and foot-fault tests. At 7 days after injection, the contralateral removal time was three times greater than the ipsilateral removal time, as well as the contralateral removal time in vehicle controls, and remained significantly elevated at 28 days. The cylinder test showed 5- and 3-fold greater forelimb asymmetry at 7 and 28 days, respectively. The percentage of foot-faults during grid exploration was greater in stroke animals than controls at both early and late time points. Importantly, the percentage of foot-faults decreased gradually in control animals as a reflection of task habituation and learning; this improvement was absent in the stroke group. Sensory function was not impaired, with normal contact times in adhesive removal test, normal turning preference in corner test, and normal contralateral thermal sensitivity in Hargreaves test. Spontaneous exploratory activity in open field test also did not differ between the groups, ruling out behavioral confounders linked to reduced locomotion. Histopathology Four weeks after ET-1 injection, the internal capsule infarct showed abundant white-matter loss (Figure 4A). The H&E staining revealed a cellular infiltrate (asterisk) at the infarct site (Figures 4B and 4C). White-matter injury was not seen in vehicle-injected control rats (Figures 4D to 4F). Immunofluorescence revealed severe loss of myelin (MBP) and axons (NF200) within the infarct (Figures 5A and 5B); some NF200 immunoreactivity was still present in the infarct possibly due to residual antigen from degenerated fibers.21 The areas of myelin and axonal loss were congruent (Figure 5C), and there was an abrupt demarcation
Figure 4. Tissue outcome 1 month after posterior internal capsule stroke. (A) White-matter infarct (arrow) 28 days after endothelin-1 injection revealed by LFB-CV staining. (B) Hematoxylin and eosin (H&E)-stained coronal section showing internal capsule lesion and high cellularity (asterisk) at the level of the infarct site. (C) High magnification image from the region marked with the asterisk reveals widespread cellular infiltration. (D–F) Absence of white-matter damage and cellular infiltration after vehicle injection. Histology was similar for all investigated animals (n = 9/group). Scale bars: 1 mm (A, B, D, and E), 0.1 mm (C and F). LFB, Luxol fast blue. © 2015 ISCBFM
Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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982 between the infarct and surrounding intact tissue at this late time point (Figure 5D). However, axonal density appeared to be reduced in all animals even in the peri-infarct tissue. This may be due to Wallerian degeneration slowly developing over days to weeks, or there may be slow, delayed, and selective axonal loss in the peri-infarct region where ischemia was milder (i.e., penumbra). Vehicle injection did not result in myelin and axonal changes in the internal capsule (Figures 5E to 5H).
DISCUSSION Here, we show that a highly focal white matter infarct in the posterior limb of internal capsule leads to pronounced and puremotor deficits that persist for at least 1 month in rats. The infarct location, lasting motor deficits, and lack of detectable sensory impairments observed in the present model resemble the typical human pure-motor syndrome associated with lacunar infarcts of the internal capsule. Therefore, stereotactic ET-1 injection is a
Figure 5. Myelin and axonal loss in the internal capsule after stroke. Immunofluorescence stainings for (A) myelin basic protein (MBP) and (B) neurofilament-200 (NF200) reveal myelin and axonal degeneration in the internal capsule (arrows) 1 month after stroke. (C) Triple fluorescence image showing congruent loss of MBP and NF200 immunoreactive fibers in the infarct core (arrow), and high cellularity detected with Hoechst 33342 (nuclei, blue). (D) High magnification image from the region marked with the asterisk in (B) showing axonal morphology in the periinfarct internal capsule and degenerated fibers in the infarct. (E–H) Normal white-matter morphology in the internal capsule after vehicle injection. Histology was similar for all investigated animals (n = 9/group). Scale bars: 0.5 mm (A–C and E–G), 0.05 mm (D and H). Journal of Cerebral Blood Flow & Metabolism (2015), 977 – 984
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Motor deficit after capsular stroke in rats F Blasi et al
983 suitable model to investigate the long-term functional outcome after focal ischemic white-matter capsular lesions. Although a significant portion of ischemic strokes involves the white matter, experimental modeling of focal white-matter injury has lagged behind large artery occlusion models. This has been in part because selective targeting of small volumes of white matter in rodent brain is technically challenging. Only about 15% to 20% of rodent brain volume is white matter, compared with 50% in human brain.8 Moreover, vascular anatomy is not permissive to selectively interrupt blood supply to white matter in rodents. Internal capsule is perfused by the anterior choroidal artery and the lenticulostriate branches of the middle cerebral artery in both human and rodents.22 Although selective occlusion of the anterior choroidal artery in pigs has created small infarcts mainly involving the internal capsule,23 in rodents infarcts have also involved thalamus and hypothalamus, thereby limiting the usefulness of this approach.24 Global hypoperfusion (e.g., bilateral carotid occlusion in rats25 or stenosis in mice26) does lead to diffuse blood flow reduction, white-matter rarefaction, and cumulative ischemic damage, but lesions mimic more chronic progressive leukoaraiosis rather than sudden-onset focal ischemic whitematter infarction. Photothrombosis has recently been used to target the internal capsule causing motor deficits lasting at least 3 weeks.27 However, sensory function was not examined in this study, and lesions also involved the entopeduncular nucleus (corresponding to the human globus pallidus internus) that may lead to motor deficits and were adjacent to the optic tract that may cause vision defects. Moreover, mechanisms of photothrombotic injury may involve more than just tissue ischemia. Finally, selective white-matter lesions can also be produced by focal injection of toxins targeting oligodendrocytes (e.g., lysolecithin injections28), but these lesions are predominantly demyelinating rather than ischemic. Compared with above, stereotactic injection of ET-1 (or other potent vasoconstrictors)29 is a unique and superior method to generate highly focal ischemia that can be strategically located to produce white-matter infarcts.10–13,30 However, lasting pure-motor deficits after a strategically located lesion have never been demonstrated. In one study,12 motor deficits were limited to a modest twofold increase in foot-faults, and sensory deficits were more prominent, possibly due to the lesion placement in anterior dorsal internal capsule. In another study in hypertensive rats,13 lesions were smaller, and motor deficits were once again very mild (cylinder test asymmetry index ~ 1.5) and limited to the forelimb (walking ladder test score ~ 5.3 on a scale of 0 worst to 6 best). Both studies followed the animals for only 2 weeks or less; therefore, persistence of the deficits was not demonstrated. Therefore, our model offers distinct and novel advantages over previously published ones. First, motor deficits were significantly more severe (e.g., threefold increase in foot-faults, cylinder test asymmetry index ~ 4), likely reflecting the optimized lesion location in the posterior internal capsule as well as larger infarct sizes (~1 mm3). That posterior lesions caused greater motor deficits in our study is in line with worse motor outcomes after strokes involving the posterior limb of internal capsule, presumably due to interruption of the dense corticospinal tract projections.3,4 Second, we showed that the deficits persisted for at least 28 days. Importantly, in the posterior injection group more than 90% of the infarct was within the internal capsule, and the small thalamic involvement did not cause sensory deficits in forelimb tactile sensitivity (contact time in adhesive removal test), vibrissae symmetry (corner test), and hind limb thermal sensitivity (Hargreaves test). The absence of mortality and good reproducibility (coefficient of variation ~ 30% for infarct volume, ~ 40% for neurologic deficits) make the model especially suitable to study older and comorbid animals,31 which are critical for clinical translation but expensive and difficult to procure. Finally, the absence of injury in other motor areas of the brain (e.g., cortex, © 2015 ISCBFM
striatum, and substantia nigra), as well as areas that may confound behavioral tests (e.g., optic nerve and tract, hippocampus), is an advantage over other models that result in white-matter injury, such as chronic forebrain hypoperfusion, chronic hypertension, intracarotid sodium laurate injection, or capsular photothrombotic lesion.9,27,32–36 Of course, ET-1 model has limitations. The mechanism of ischemia does not mimic lacunar pathophysiology, multiple vessels constrict at the same time, the duration of ischemia cannot be precisely controlled, and ET-1 targets not only endothelial and smooth muscle cells but also neurons and glia as a possible confounder.10,37 Lacunar infarcts account for 25% of all ischemic strokes,38 and are caused by occlusion of a single penetrating artery due to microatheroma or lipohyalinosis,39 although this is still debated.40 None of the existing whitematter stroke models accurately recapitulates small vessel disease and lacunar stroke mechanisms.32 For these reasons, the model is better suited to investigate recovery in chronic stages, as well as ischemic white-matter injury progression and repair after the hyperacute stage (e.g., 412 hours), which differs from gray matter given the predominantly oligodendrocyte and axon injury rather than neuronal cell bodies.9 Functional outcome is a primary end point after stroke, but repair and recovery after white-matter stroke are still poorly understood due to a lack of experimental models that yield reproducible and lasting functional deficits. Therefore, our model represents a critical improvement to facilitate preclinical investigations of novel therapeutic interventions targeting recovery after white-matter injury. DISCLOSURE/CONFLICT OF INTEREST The authors declare no conflict of interest.
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