Behavioural Brain Research 261 (2014) 202–209
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Functional assessment of sensory functions after photothrombotic stroke in the barrel field of mice Monika Liguz-Lecznar a,∗ , Renata Zakrzewska a , Katarzyna Daniszewska a , Malgorzata Kossut a,b a b
Laboratory of Neuroplasticity, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland Warsaw School of Social Science and Humanities, 19 Chodakowska St., 03-815 Warsaw, Poland
h i g h l i g h t s • • • • •
We assessed sensorimotor functions in mice after focal stroke within barrel cortex. Motor functions were unaffected. Behavioural tests revealed sensory deficits of variable duration and intensity. Basic vibrissal sensation was spared with strong and impaired with weaker stimuli. Cognitive impairments were detectable with sensory labyrinth test.
a r t i c l e
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Article history: Received 21 October 2013 Received in revised form 18 December 2013 Accepted 21 December 2013 Available online 31 December 2013 Keywords: Photothrombotic stroke Behaviour Mice Barrel cortex
a b s t r a c t Motor, sensory and cognitive deficits are common impairments observed in human stroke as well as in animal stroke models. Using a battery of behavioural tests we assessed sensorimotor deficits after photothrombotic stroke localized within or beyond cortical representation of mouse sensory vibrissae. We found restricted, modality specific behavioural consequences in the acute post-stroke period. Among incorporated tests, adhesive removal test, novelty exploration test and sensory labyrinth task were sensitive to the somatosensory cortical deficits. Injured animals explored new objects significantly longer, they also needed distinctly more time to contact and to remove the adhesive tape placed on whiskers contralateral to the infarct. Moreover, we observed that after stroke animals were unable to solve the sensory labyrinth depending only upon tactile sensation from whiskers with injured cortical representation. Spontaneous recovery could be observed within the first post-stroke week for adhesive tape removal and within 14 days for labyrinth performance. However, for the novel object exploration we did not observed the recovery for the period of 18 days after stroke. Moreover, new object exploration test performance differed between the somatosensory and visual cortical impairments. We suggest that those three tests might be valuable in assessing the usefulness of therapies designed to support brain repair after experimental stroke. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Studies of function compensation in stroke research are necessary especially for investigating the effectiveness of therapies promoting regeneration and repair. Most animal studies examined the sensorimotor deficits after global and focal ischemia [1–3]. The tests of choice should be sensitive to the site of damage, degree of injury and severity of impairment. Since loss of limb function is a common consequence of human stroke, many studies in rodent models of stroke focused on motor and sensorimotor tests, assessing motor
∗ Corresponding author. Tel.: +48 22 5892248; fax: +48 22 8225342. E-mail address: [email protected] (M. Liguz-Lecznar). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.12.027
coordination and balance (rotarod), sensory asymmetry (adhesive tape removal test) as well as the forepaw (cylinder test, forelimb placing, reaching tasks, staircase test) and hindlimb (grid walking, ledged tapered beam) functions [4]. However, sensory perception in rodents is strongly based on the vibrissal sensation. Relying only on the sensory vibrissae animals can assess the distance between objects, texture and roughness [5,6]. Our earlier studies showed that unilateral focal stroke in cortical representation of sensory whiskers in rats disturbed the performance in the gap-crossing task and functional recovery was correlated with induction of increased activity foci in a ipsilesional spared somatosensory areas, responding specifically to the vibrissal stimulation [7]. In acute post-stroke phase however, we and others observed a strong decrease in cortical activation on the lesion side, measured with 2DG incorporation
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in the barrel [7] and auditory cortex [8]. Also, Pappius [9] observed a decreased blood flow as early as 4 h after the freezing lesion. Additionally, brain infarct causes inflammatory responses beginning within a few hours after stroke at the injury site as well as in the surrounding area [10–13], which can impair cortical function [14]. Driven by the need for better understanding of mechanisms of stroke-related injuries and recovery, we decided to further investigate the acute behavioural outcome after focal photothrombotic stroke within sensory vibrissae representation (barrel cortex) in mice. Animals were tested for their sensorimotor functions. The object of the present study was to determine which functions are impaired in the acute post-stroke phase and which tests are sensitive for the functional impairment of vibrissal system. 2. Materials and methods 2.1. Animals Experiments were performed on 43 young-adult (8–10 weeksold) C57BL/6 J mice. The animals were kept in a temperaturecontrolled room (20 ◦ C) with a 12/12 h light/dark cycle and had free access to food and water unless otherwise stated. All work with animals was carried out in accordance with the European Communities Council Directive (86/609/EEC) and was approved by the Animal Care and Use Committee of the Polish Academy of Sciences. From those animals we have created following groups: Animals that were tested in all incorporated tests apart from sensory labirynth test: • • • •
control naive: n = 5 sham-operated: n = 8 stroke within barrel cortex: n = 9 stroke beyond barel cortex: n = 6.
Not all animals were incorporated in all tests-see “n” value for particular tests. Animals that were tested in sensory labirynth test: • sham-operated: n = 8 • stroke within barrel cortex and unilateraly removed whiskers: n = 9. 2.2. Surgical procedures Unilateral cortical lesions were induced photochemically. We modified the method introduced originally for rats by Watson et al. [15]. Before surgery, the animals were anesthetized with 3% isoflurane/air (Baxter, Lessiness, Belgium). The skin above the skull was incised and a fiber-optic bundle mounted on a cold light source ( = 1.5 mm, wavelength 560 nm, aperture B2, 2750 K, KL 1500 LCD, Schott, Germany), was placed over the right hemisphere with a focus at 1.4 mm posterior to bregma and 3 mm lateral to the midline (barrel cortex). Photosensitive dye Bengal Rose (disodium tetraiodo-tetrachlorofluoroscein, Sigma Aldrich Chemie) was injected into the lateral tail vain (100 l of the dye; 10 mg/ml) and immediately after the injection we started focal illumination of the skull, that lasted 20 min. Placement of the light beam, the light intensity and the light aperture were the same for all animals. After the induction of thrombosis, the incisions were sutured and animals were returned to their home cages. As controls we used sham-operated animals, which were subjected to the same procedure except for light irradiation. Second control group consisted of animals with stroke placed in visual cortex: light source focused at 2.5 mm posterior to bregma and 2 mm lateral
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to the midline (area of visual cortex). If in the particular test there were no differences between the two control groups the data were pooled and presented as a control group. 2.3. Behavioural tests 2.3.1. Activity and exploratory behaviour Activity and exploratory behaviour of mice were observed in open field for a 10-min period. Time of activity including motion, rearing, grooming versus time of freezing was measured. Data are presented as a % of activity before the operation. 2.3.2. Rotarod This test is used to assess sensorimotor coordination and balance [3]. Animals were placed on a rotating rod and the speed of rotation was gradually increased. Ability of mice to remain on the rod was measured. We have used TSE RotaRod (Advance System) with rotating speed from 7.5 to 60 rpm, acceleration every 3 s with constant speed for 2 s. Preoperative training was performed for 2 days with 4 daily trials, with three last trials serving as the preoperative baseline. Postoperative testing was performed 1 and 7 days after stroke, three trials per day and the mean was used for analysis. 2.3.3. Nest building test Mice were individually housed overnight, with food, water and sawdust. A standard piece of paper towel (23 × 23 cm), from which the mice could make nests, was placed in each cage. Nests usually consisted of a low mound of sawdust with a crater in the top, surrounded by shredded cotton. They were scored like described earlier [16] according to the following scale: 0—no visible piling of sawdust, no shredded cotton; 1—sawdust mound and crater alone, no shredded cotton; 2—sawdust mound and crater, with shredded cotton gathered around and in the crater to form a cup-shaped nest; 3—the shredded cotton forms a ball-shaped nest covering the mouse. Animals were tested before and one week after the stroke. 2.3.4. Whisker nuisance task (whisker response) Testing involved unilateral manual stimulation of the whiskers of both mystical pads with a smooth brush. Each animal was tested in 10 probes (5 for each whisker pad) for each time-point: before the stroke, 1, 7 and 14 days after the stroke. Animals were tested individually in their home cages. For each animal, observations were made regarding the predominant observed behaviour. Each behaviour expressed in response to whisker stimulation has assigned following scores depending on degree of expression: 0—lack of reaction; 1—moderate reaction (slight whisker, head or body movement), 2—vigorous reaction (rapid whisker or head movement, body turn, grooming). Scores for each side were summed up and averaged. 2.3.5. Adhesive removal test The response to sensory stimuli was measured with adhesive removal test that has been first adapted to rats about a quarter of century ago [17] and consists of adhesive tapes applied to different parts of the animal body (forelimbs, hindlimbs or snout). It measures sensory functions, sensory neglect and motor functions independently for the left and the right side and have been proved to be one of the most efficient test to detect very small stroke related sensorimotor impairments. Time to contact and time to remove the stimulus separate out sensory and motor deficits, respectively [4]. We have modified the test to detect potential impairments of vibrissae sensation. Small adhesive tape square (2 × 2 mm) was placed on right or left C2 or C3 vibrissae of the mouse. The order of placement of the adhesive (right or left) was alternated between each animal and each session. The mouse was then placed in a home cage, and the times to contact and to remove each adhesive tape
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were measured, with a limit of 60 s. Four trials per testing day, two for each side for each animal were performed. Mice were tested before surgery and then 1, 7, 10 and 14 days after stroke induction.
(control group, Fig. 1A and C) and with intact subcortical structures were taken into further analysis. 3.2. Activity and exploratory behaviour
2.3.6. New object exploration Animals were tested for exploration of new objects in their home cages. All animals were presented with the same set of objects and none of the objects was presented twice within one session. Testing consisted of four 2-min. trials, with a 1-min. intertrial interval between each trial. Test was conducted before the stroke, 1, 4, 6, 10 and 18 days after the stroke. All procedures were conducted during the light cycle of the animal between 9 a.m. and 2 p.m. To control for possible odour cues the objects were cleaned with a 10% ethanol solution at the end of each trial. In each trial, the time to approach the object as well as the number of contacts and the total time of exploration of each object were measured using a stopwatch. Exploration was defined as direct contact of the whiskers, nose or front paws with the object. 2.3.7. Sensory labyrinth For sensory labyrinth test all the whiskers on one side of the snout were systematically trimmed (the other side remained intact) and mice were trained to pass through a labyrinth (30 × 40 cm) in the darkness using only tactile cues. Animals were rewarded with the food, which was placed always in the same location of the labyrinth. To increase motivation, food availability was restricted to 70% of daily rations during the whole period of the training and test. To control for possible odour cues, the labyrinth was cleaned with a 10% ethanol solution at the end of each trial. In the training phase each mice was placed in the labyrinth 4 times a day and this phase lasted until all animals were able to pass the labyrinth in 1 min. without returning to the start point. Than, stroke was induced within the barrel field contralateral to the intact whiskers. In the test phase each animal was tested twice at each time point: before the stroke, 1, 2, 7, 9, 11, 14, 15, 16, 17, and 18 days after ischemia. The average time of reaching the goal was then compared for sham operated and stroked animals.
There were no differences in the spontaneous motor activity between control (healthy and sham-operated pooled, n = 11) and stroke animals, regardless of the localization of the infarct in the barrel (n = 9) or visual cortex (n = 6), respectively, at the time of observation: 1 day (95.4% ± 1.07%, 93.57% ± 3.26%, and 89% ± 2.3%); 7 days (96.3% ± 1.6%, 93.73% ± 1.39% and 90.5% ± 5.5%) or 14 days (95.5% ± 1.8%, 97.28% ± 1.2% and 92.3% ± 7.85%) after stroke (ANOVA F(3,32) = 2.17, P = 0.1). Fig. 2A presents results of activity measurements for control and ischemic animals (group with the stroke within the barrel cortex). 3.3. Rotarod assessment of sensorimotor deficits Analysis of variance revealed no significant effect of time postlesion on rotarod performance (F(2,21) = 0.29, P = 0.75, Fig. 2B). The time spent on the rotarod by sham-operated (n = 6) and operated (stroke in the barrel cortex, n = 7) animals was comparable before and after the stroke: sham-operated—155.4 ± 18.44 s before the operation; 160 ± 10.32 s 1 day after and 166 ± 13.22 s 7 days after operation; operated animals: 168.4 ± 14.38 s before the stroke; 156 ± 9.23 s 1 day after and 168 ± 14.37 s 7 days after operation. There were no differences between mice with stroke in the barrel and visual cortex (data not shown). 3.4. Nest building test During analysis of the abilities of nest building, which is one of the species typical behaviour in mice, we failed to find a significant overall influence of stroke on nesting behaviour (Fig. 2C). There was no significant interaction between the infarct placement and paper towel nest construction (ANOVA P > 0.05, n = 24). 3.5. Whisker nuisance task (whisker response)
2.4. Verification of the infarct localization After completion of all tests animals were sacrificed, the brains were removed, frozen and cut on the cryostat. Representative sections from each brain were stained with cresyl violet to visualize the infarct localization and the longest axis of infarct was measured using a Nikon Eclipse 80i microscope equipped with a digital camera (Evolution VF; MediaCybernetics). 2.5. Statistical analysis The data were incorporated into a repeated measures ANOVA for parametric or Kruskal–Wallis for non-parametric analysis of variance. Specific effects were evaluated by Tukey or Dunn’s multiple comparison post-test, P < 0.05 was considered statistically significant. 3. Results 3.1. Size and localization of the infarct Lesions were about 2 mm in diameter (from 1.89 ± 0.6 to 2.33 ± 0.9 mm), and spanned all cortical layers leaving the subcortical structures intact. Only animals with verified size and localization of stroke embracing whole barrel cortex (experimental groups, Fig. 1A and B) or localized entirely beyond barrel cortex
A longitudinal evaluation of the uninjured sham animals (n = 8) and stroke animals irrespective of the stroke placement (n = 11) tested prior to, 1 day, week and 2 weeks after a sham injury did not show any aberrant behaviours in response to whisker stimulation (Fig. 2D). Whisker nuisance scores obtained by each particular group of tested animals were no higher than 11 at any time point, of a maximum 20 score. There were no significant differences between analyzed time points in injured animals (ANOVA, P > 0.05). Whisker Nuisance Task did not differentiate sham from ischemic mice over 2 weeks post-injury. 3.6. Adhesive removal test To remove the tape, animals raised both forelimbs towards their face and swiped off the stimulus with both forepaws. Typically, uninjured mice made contact and simultaneously removed the tape within 10 s. We did not observe significant differences between control (8.07 ± 0.65 s, n = 5) and sham operated animals (9.1 ± 0.98 s, n = 8) or stroke animals when adhesive tape was placed on whiskers ipsilateral to stroke (8.5 ± 0.63 s, n = 9). When stimulus was placed on whiskers contralateral to the infarct, one day after stroke the animals needed significantly more time to remove the tape than before the stroke (22.78 ± 3.4 s vs. 8.07 ± 0.65 s; ANOVA, F(4,44) = 15.54, P > 0.001, n = 9, Fig. 3D). However, there was no lag between contact and removing of stimulus. Seven days after stroke and at later post-stroke times there was no evidence of significant
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Fig. 1. (A) Schematic localization of photothrombotic infarct in the vicinity or within the barrel cortex, a cortical representation of sensory vibrissae and picture showing the infarcted brain. (B) Representative tangential sections stained with cresyl violet from control (left) and stroked (right) hemisphere showing the infarct within barrel cortex. (A–E) Rows of barrels. x-marking holes enabling alignment of consecutive slices. (C) Representative tangential and coronal sections stained with cresyl violet presenting localization of infarct within visual cortex beside the barrel field (BF) and showing that subcortical structures were intact.
extension of stimulus removal time in stroke animals (7 days poststroke: 6.78 ± 0.74 s; 10 days post-stroke: 7.94 ± 0.67 s and 14 days post-stroke: 8.67 ± 1.49; Fig. 3D). Initial contact with the stimulus went hand in hand with its removal which speaks for the absence of motor impairments. 3.7. New object exploration During the observation of stroked mice exploring a new object we noted significant effects of stroke localization on the animals’ behaviour. Mice with infarct localized in the barrel cortex spent significantly longer time exploring objects with vibrissae than control animals (17.67 ± 1.5 s vs. 6.22 ± 0.57 s) and that effect persisted for the whole period of post-stroke observation (ANOVA, F(5,53) = 2.45, P = 0.001, n = 9) (Fig. 3A). Detailed results for particular post-stroke days are summarized in Table 1A. In this group of animals we did not observed any significant interaction between stroke and the time the animals needed to approach the object (ANOVA, F(5,53) = 0.5, P > 0.05) or in the number of contacts with the objects (ANOVA, F(5,53) = 2.57, P > 0.05) (Table 1A). In the group of mice with infarct outside of the barrel cortex, embracing part of visual cortex, a significant interaction between the stroke and the time of approaching the object could be observed (ANOVA, F(5,25) = 6.4, P > 0.02, n = 6). Animals with stroke needed more time to approach the object (10 ± 2.72 s before the stroke vs. 20.13 ± 3.6 s 1 day after stroke). The observed effect was transient and after one week of recovery was no longer observed (16.75 ± 9.5 s) (Table 1B, Fig. 3B). This group of mice did not show any effect of stroke on the time spent on the object exploration or on the number of contacts with objects (ANOVA, F(5,33) = 2.04, P > 0.05, Table 1B). 3.8. Sensory labyrinth We have observed a considerable influence of stroke upon performance in the labyrinth. Mice with stroke in the vibrissae representation were unable to complete the task, which they had learned before the stroke. Before the stroke the passage through
labyrinth took them in average19 ± 3.8 s whereas one day after the stroke they needed 64.3 ± 26.6 s in average, which was more than three times longer (ANOVA, F(10,66) = 13.41, P = 0.02, n = 8) (Fig. 3C). For sham-operated animals the passage time was similar for the whole tested period and amounted 13.9 ± 3 s (Fig. 3C, n = 6). After 14 days of recovery stroke animals re-learned to navigate straightforward to the finish point in the labyrinth with average time of 13 ± 1.5 s. Detailed results for consecutive days are presented in the table (Table 2).
4. Discussion We assessed sensorimotor functions of mice after the focal photothrombotic stroke. We found that unilateral photothrombotic injury within or in the neighbourhood of barrel cortex, a part of primary somatosensory cortex, produce restricted, modality specific behavioural consequences in the acute post-stroke period. Our earlier data have shown that in rats and mice, vicinity of infarct impairs functional cortical plasticity induced by sensory deprivation of vibrissae. [14,18,19]. Here, using a battery of behavioural tests (global and modality specific) we evaluated the sensory functions in the acute post-ischemic phase, to establish which functions might be the most susceptible to the detrimental effects of stroke. Earlier, we and others showed that ischemic injury of the whisker representation impairs performance of gap-crossing task and this effect is still present more than one month after stroke [7,20,21]. We did not observed any differences in the normal exploratory behaviour and activity between healthy and operated animals even as early as one day after surgery. That implied that animals did not suffer from post-surgery pain or depression that could interfere the performance of behavioural test we used. Accelerated rotarod was used to assess the motor coordination and balance alterations. Earlier publications reported the impairment of rotarod performance after photothrombotic stroke, but the infarct embraced limbs representation in the sensorimotor cortex [22,23]. Here, the infarct was localized in the proximity of forepaw representation, but it did not interfere in its functioning, since
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Fig. 2. (A) Chart showing results of activity and exploratory behaviour analysis. Data are presented as a percent of pre-ischaemic activity. No differences were observed during two weeks after stroke between sham-operated and stroke groups. Data represent mean ± SEM. (B) Sensorimotor coordination and balance remained unaffected during the first post-stroke week. Chart presents the time during which animals were able to stay on the rod (s). Data represent mean ± SEM. (C) Nesting behaviour was not significantly impaired after stroke. There was no difference between sham-operated and stroke animals. Chart present points average points given by two unrelated observers. Data represent mean ± SEM. (D) Results of whisker nuisance test showing no significant differences in whiskers reactivity to the tactile stimulus between sham-operated and stroke animals during two post-stroke weeks. Chart presents an average points given by two unrelated observers. Data represent mean ± SEM.
ischaemic animals did not differ from the control group according to the rotarod performance. We have assessed also the nesting behaviour, which is one of the maintenance related behaviours, displayed by both sexes in parental and non-parental contexts and is controlled by levels of arousal and motivation [24]. Nest building was impaired by medial prefrontal and hippocampal lesions in rats and hamsters but not in mice [15,25–27] suggesting that this result might be species specific. Our mice presented normal nesting activity implying proper functioning of hippocampus and prefrontal cortical areas.
To estimate the vibrissae-mediated perception we have conducted several somatosensory specific tests. Whisker Nuisance test allows to assess the reactivity to the tactile stimulus and was effective in detecting post-traumatic morbidity following the diffuse brain injury in rats. After brain injury animals were overactive to the tactile whisker stimulation what was related to the stress response [28–30]. In our experiments we did not detect any change in animals’ reactivity to the whisker-delivered stimulus after ischemia irrespective of the infarct localization. On the other hand, in the adhesive removal test early after stroke we have observed
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Fig. 3. (A) Chart showing results of new objects exploration test in animals with infarct within barrel cortex. After stroke animals spent significantly more time exploring new objects than before ischaemia. The effect sustained throughout whole observation period (18 days after stroke). Data represent mean ± SEM. * P < 0.05; ** P < 0.01. (B) Chart showing results of new objects exploration test in animals with infarct within visual cortex. One day after stroke animals needed significantly more time to achieve object than before ischaemia. This effect could not be observed a week after stroke. Data represent mean ± SEM. * P < 0.05. (C) Results of sensory labyrinth test showing the impairment of labyrinth performance one day after stroke and recovery of function after 15th post-stroke day. Sham-operated animals were unaffected. Data represent mean ± SEM. ** P < 0.01. (D) Results of adhesive removal test. Chart represents the time needed to contact and remove the stimulus from whiskers contralateral to the injured barrel cortex. Sensory deficits could be observed one day after stroke with the recovery within the first post-stroke week. Data represent mean ± SEM. *** P < 0.001. Table 1 Results for novel object exploration task for animals with stroke within the barrel cortex (A) and beside the barrel cortex within the area of visual cortex (B). (A). Parameter/time (s); average ± SEM
Before stroke
1 day
4 days
6 days
10 days
18 days
Time to approach Number of contacts Time of exploration
32.44 ± 5.3 3 ± 0.52 6.22 ± 0.57
23.44 ± 11.58 5.22 ± 0.74 17.67 ± 1.5*
33.11 ± 9.19 4 ± 0.66 20.67 ± 3.4**
43.22 ± 7.94 3.4 ± 0.55 23.11 ± 6.6**
31.11 ± 6.75 5.3 ± 0.62 25.89 ± 6.5*
34.44 ± 11.29 6 ± 1.15 19.78 ± 3.1*
(B) Parameter/time (s); average ± SEM Time to approach Number of contacts Time of exploration * * **
Before stroke 10 ± 1.7 3.5 ± 0.42 8.3 ± 2.48
1 day 21.3 ± 2.7* 4.2 ± 0.7 9.3 ± 2.4
Significant difference with the result before the ischemia; 1 day, 4 days, etc.—days after stroke. Data represent mean ± SEM. P < 0.05. P < 0.01.
7 days 16.83 ± 3.06 4.1 ± 0.4 11.3 ± 2.3
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Table 2 Time to complete the sensory labyrinth task for sham-operated and ischemic animals.
*
Group/time (s); average ± SEM
Before stroke
1 day
2 days
7 days
9 days
11 days
14 days
15 days
16 days
17 days
18 days
Sham operated Ishemic
13.5 ± 1.1 19.3 ± 2.4
14.0 ± 1.0 64.3 ± 6.9**
15.5 ± 1.5 66.3 ± 5.7**
12.5 ± 0.9 73.2 ± 11.1***
11.5 ± 1.6 57.2 ± 7.0**
15.0 ± 1.4 52.0 ± 2.4**
14.0 ± 1.1 67.2 ± 5.8***
13.0 ± 1.7 9.2 ± 0.6
18.0 ± 1.7 21.3 ± 2.3
11.0 ± 1.2 14.8 ± 1.1
14.5 ± 1.4 13.0 ± 1.1
Significant difference with the result before the ischemia; 1 day, 2 days, etc.—days after stroke. Data represent mean ± SEM. ** P < 0.01. *** P < 0.001.
significant stroke-related impairments illustrating the infarctinduced sensory deficit. In the whisker nuisance test however, tactile stimulation is much stronger and spared subcortical vibrissal centres may be sufficient for the stimulus processing. Correlations between cortical lesions and ischemia-induced behavioural impairments in the adhesive removal test have been well investigated in the literature and many authors have demonstrated a close correlation between contralateral removal latencies on this task and changes in the forelimb cortex following transient MCAO and photothrombosis [31–34]. In those experiments however, observed impairments lasted at least 3 weeks, whereas in our investigations sensory deficits were visible only early after stroke with recovery within the first post-stroke week. This suggests rapid activation of compensatory mechanism aiming at retaining of the basic tactile sensation. Novel object exploration examined the reactivity of ischemic animals to novelty, and the basic premise of this test is that animals have the natural curiosity which impels them to explore novel environments. The aim of the test was to estimate if unilateral ischemic disruption of barrel cortex will influence the exploratory behaviour of mice. Our data indicate that animals with the stroke within barrel cortex explored objects for a significantly longer time than the control group. The time of object approaching and the number of contacts with objects did not change which suggests unaffected reactivity of injured animals. Thus, we suggest that mice needed more time to collect all necessary information about each object. The observed effect persisted through the entire observation period and we did not observed behavioural recovery. Although, based on the Whisker nuisance Test results it may seem that touch sensation is not impaired after ischemic injury of barrel cortex, we need to note that here, animals need to collect more complex information about size, structure and texture of the object and integrate them in the higher brain areas. Also, those two task differ in nature, with Nuisance test relying on involuntary (passive) and Novelty test on voluntary (active) touch perception. The novel object exploration test turned to be sensitive to the stroke localization. Mice with unilateral infarct beside the barrel field but embracing significant part of visual cortex needed more time to approach the object while the number of contacts and time of exploration remained unchanged. Since this effect was transient and could be observed only one day after stroke, we suggest that it was caused by some disturbances of visual symmetry, which could be easily overcome when animals accommodate to changed visual perception. Cognitive ability was checked with the sensory labyrinth test. It involves spatial and working memory assessment and tactile navigation through the labyrinth in the dark requires sensory information from the whiskers similarly like during subterranean burrows behaviour [35,36]. Our labyrinth task was conducted in the dark since to avoid usage of visual cues [37,38]. Stroke within the representation of intact whiskers resulted in initial inability to solve the labyrinth. However, we observed the complete recovery after 14 days. Times that animals needed to learn and than re-learn the labyrinth task after stroke were comparable what suggests that memory about this task performance was located in
the destroyed cortex and acquisition of memory had to start from the beginning. Earlier studies that have examined the behavioural results of barrel cortex infarction presented diverse effects on the cognitive tests performance, but above all on the recovery and return of function. Guic-Robles and colleagues have shown that roughness discrimination task is barrel cortex-dependent, and after its bilateral lesion no behavioural recovery can be observed [39]. In other study Hurwitz et al. [40], have tested animals in the Gap Crossing Test and showed robust behavioural deficits, but with 80% recovery. Then, Pazos et al. [41] have demonstrated that behavioural recovery of rats after photothrombosis tested in T-maze is not dependent upon the remaining somatosensory cortical tissue. Moreover, they suggested that neither the contralateral somatosensory cortex nor the vibrissal representation within ipsilateral secondary somatosensory cortex play a critical role in the observed recovery process. Our studies does not allow us to take a stand on this dispute, however, since in our investigations both, contralateral primary somatosensory cortex and secondary somatosensory cortices remained intact, they could possibly take part in restoration of behavioural functions after ischemia. Barrel neurons are influenced also by the whiskers on the ipsilateral side of the face and this response is mediated via callosal connections [42,43], which are supposed to provide at least part of the pathway for signaling the presence of an infarct. Behavioural studies have shown that tactile learning occurring in one barrel cortex leads to significant transfer to the homologous barrel on the other side [44]. Also, secondary somatosensory cortex (SII), which contains the representation of facial vibrissae and is reciprocally connected with the ipsilateral primary somatosensory cortex [45,46] could possibly support the post-stroke functional recovery. It is postulated that SII receives direct input from the thalamus independently from SI [47] and we have shown that the response of SII cortex to vibrissal input was modified bilaterally after classical conditioning which involved unilateral tactile stimulation [48]. Thus, after unilateral injury of primary barrel cortex, information acquired with the involvement of uninjured subcortical structures might be directed towards the spared areas of ipsilateral cortex or to the contralateral homotopic cortex.
5. Conclusions Photothrombotic stroke within cortical representation of sensory vibrissae in its acute phase induced sensory deficits detectable with specific behavioural tests. Basic sensation of whisker stimulation was spared with strong stimulus (whisker nuisance test) while impaired when weaker tactile stimuli (adhesive removal test) were applied. Cognitive sensory abilities were impaired as revealed by sensory labyrinth task performance, however animals retained abilities of task re-learning. Pronounced and long-lasting sensory deficits were visible when animals was confronted with new environmental cues that had to be explored with vibrissae (new object exploration test). For this test no functional recovery could be observed.
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Research report
Functional assessment of sensory functions after photothrombotic stroke in the barrel field of mice Monika Liguz-Lecznar a,∗ , Renata Zakrzewska a , Katarzyna Daniszewska a , Malgorzata Kossut a,b a b
Laboratory of Neuroplasticity, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland Warsaw School of Social Science and Humanities, 19 Chodakowska St., 03-815 Warsaw, Poland
h i g h l i g h t s • • • • •
We assessed sensorimotor functions in mice after focal stroke within barrel cortex. Motor functions were unaffected. Behavioural tests revealed sensory deficits of variable duration and intensity. Basic vibrissal sensation was spared with strong and impaired with weaker stimuli. Cognitive impairments were detectable with sensory labyrinth test.
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 18 December 2013 Accepted 21 December 2013 Available online 31 December 2013 Keywords: Photothrombotic stroke Behaviour Mice Barrel cortex
a b s t r a c t Motor, sensory and cognitive deficits are common impairments observed in human stroke as well as in animal stroke models. Using a battery of behavioural tests we assessed sensorimotor deficits after photothrombotic stroke localized within or beyond cortical representation of mouse sensory vibrissae. We found restricted, modality specific behavioural consequences in the acute post-stroke period. Among incorporated tests, adhesive removal test, novelty exploration test and sensory labyrinth task were sensitive to the somatosensory cortical deficits. Injured animals explored new objects significantly longer, they also needed distinctly more time to contact and to remove the adhesive tape placed on whiskers contralateral to the infarct. Moreover, we observed that after stroke animals were unable to solve the sensory labyrinth depending only upon tactile sensation from whiskers with injured cortical representation. Spontaneous recovery could be observed within the first post-stroke week for adhesive tape removal and within 14 days for labyrinth performance. However, for the novel object exploration we did not observed the recovery for the period of 18 days after stroke. Moreover, new object exploration test performance differed between the somatosensory and visual cortical impairments. We suggest that those three tests might be valuable in assessing the usefulness of therapies designed to support brain repair after experimental stroke. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Studies of function compensation in stroke research are necessary especially for investigating the effectiveness of therapies promoting regeneration and repair. Most animal studies examined the sensorimotor deficits after global and focal ischemia [1–3]. The tests of choice should be sensitive to the site of damage, degree of injury and severity of impairment. Since loss of limb function is a common consequence of human stroke, many studies in rodent models of stroke focused on motor and sensorimotor tests, assessing motor
∗ Corresponding author. Tel.: +48 22 5892248; fax: +48 22 8225342. E-mail address: [email protected] (M. Liguz-Lecznar). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.12.027
coordination and balance (rotarod), sensory asymmetry (adhesive tape removal test) as well as the forepaw (cylinder test, forelimb placing, reaching tasks, staircase test) and hindlimb (grid walking, ledged tapered beam) functions [4]. However, sensory perception in rodents is strongly based on the vibrissal sensation. Relying only on the sensory vibrissae animals can assess the distance between objects, texture and roughness [5,6]. Our earlier studies showed that unilateral focal stroke in cortical representation of sensory whiskers in rats disturbed the performance in the gap-crossing task and functional recovery was correlated with induction of increased activity foci in a ipsilesional spared somatosensory areas, responding specifically to the vibrissal stimulation [7]. In acute post-stroke phase however, we and others observed a strong decrease in cortical activation on the lesion side, measured with 2DG incorporation
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in the barrel [7] and auditory cortex [8]. Also, Pappius [9] observed a decreased blood flow as early as 4 h after the freezing lesion. Additionally, brain infarct causes inflammatory responses beginning within a few hours after stroke at the injury site as well as in the surrounding area [10–13], which can impair cortical function [14]. Driven by the need for better understanding of mechanisms of stroke-related injuries and recovery, we decided to further investigate the acute behavioural outcome after focal photothrombotic stroke within sensory vibrissae representation (barrel cortex) in mice. Animals were tested for their sensorimotor functions. The object of the present study was to determine which functions are impaired in the acute post-stroke phase and which tests are sensitive for the functional impairment of vibrissal system. 2. Materials and methods 2.1. Animals Experiments were performed on 43 young-adult (8–10 weeksold) C57BL/6 J mice. The animals were kept in a temperaturecontrolled room (20 ◦ C) with a 12/12 h light/dark cycle and had free access to food and water unless otherwise stated. All work with animals was carried out in accordance with the European Communities Council Directive (86/609/EEC) and was approved by the Animal Care and Use Committee of the Polish Academy of Sciences. From those animals we have created following groups: Animals that were tested in all incorporated tests apart from sensory labirynth test: • • • •
control naive: n = 5 sham-operated: n = 8 stroke within barrel cortex: n = 9 stroke beyond barel cortex: n = 6.
Not all animals were incorporated in all tests-see “n” value for particular tests. Animals that were tested in sensory labirynth test: • sham-operated: n = 8 • stroke within barrel cortex and unilateraly removed whiskers: n = 9. 2.2. Surgical procedures Unilateral cortical lesions were induced photochemically. We modified the method introduced originally for rats by Watson et al. [15]. Before surgery, the animals were anesthetized with 3% isoflurane/air (Baxter, Lessiness, Belgium). The skin above the skull was incised and a fiber-optic bundle mounted on a cold light source ( = 1.5 mm, wavelength 560 nm, aperture B2, 2750 K, KL 1500 LCD, Schott, Germany), was placed over the right hemisphere with a focus at 1.4 mm posterior to bregma and 3 mm lateral to the midline (barrel cortex). Photosensitive dye Bengal Rose (disodium tetraiodo-tetrachlorofluoroscein, Sigma Aldrich Chemie) was injected into the lateral tail vain (100 l of the dye; 10 mg/ml) and immediately after the injection we started focal illumination of the skull, that lasted 20 min. Placement of the light beam, the light intensity and the light aperture were the same for all animals. After the induction of thrombosis, the incisions were sutured and animals were returned to their home cages. As controls we used sham-operated animals, which were subjected to the same procedure except for light irradiation. Second control group consisted of animals with stroke placed in visual cortex: light source focused at 2.5 mm posterior to bregma and 2 mm lateral
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to the midline (area of visual cortex). If in the particular test there were no differences between the two control groups the data were pooled and presented as a control group. 2.3. Behavioural tests 2.3.1. Activity and exploratory behaviour Activity and exploratory behaviour of mice were observed in open field for a 10-min period. Time of activity including motion, rearing, grooming versus time of freezing was measured. Data are presented as a % of activity before the operation. 2.3.2. Rotarod This test is used to assess sensorimotor coordination and balance [3]. Animals were placed on a rotating rod and the speed of rotation was gradually increased. Ability of mice to remain on the rod was measured. We have used TSE RotaRod (Advance System) with rotating speed from 7.5 to 60 rpm, acceleration every 3 s with constant speed for 2 s. Preoperative training was performed for 2 days with 4 daily trials, with three last trials serving as the preoperative baseline. Postoperative testing was performed 1 and 7 days after stroke, three trials per day and the mean was used for analysis. 2.3.3. Nest building test Mice were individually housed overnight, with food, water and sawdust. A standard piece of paper towel (23 × 23 cm), from which the mice could make nests, was placed in each cage. Nests usually consisted of a low mound of sawdust with a crater in the top, surrounded by shredded cotton. They were scored like described earlier [16] according to the following scale: 0—no visible piling of sawdust, no shredded cotton; 1—sawdust mound and crater alone, no shredded cotton; 2—sawdust mound and crater, with shredded cotton gathered around and in the crater to form a cup-shaped nest; 3—the shredded cotton forms a ball-shaped nest covering the mouse. Animals were tested before and one week after the stroke. 2.3.4. Whisker nuisance task (whisker response) Testing involved unilateral manual stimulation of the whiskers of both mystical pads with a smooth brush. Each animal was tested in 10 probes (5 for each whisker pad) for each time-point: before the stroke, 1, 7 and 14 days after the stroke. Animals were tested individually in their home cages. For each animal, observations were made regarding the predominant observed behaviour. Each behaviour expressed in response to whisker stimulation has assigned following scores depending on degree of expression: 0—lack of reaction; 1—moderate reaction (slight whisker, head or body movement), 2—vigorous reaction (rapid whisker or head movement, body turn, grooming). Scores for each side were summed up and averaged. 2.3.5. Adhesive removal test The response to sensory stimuli was measured with adhesive removal test that has been first adapted to rats about a quarter of century ago [17] and consists of adhesive tapes applied to different parts of the animal body (forelimbs, hindlimbs or snout). It measures sensory functions, sensory neglect and motor functions independently for the left and the right side and have been proved to be one of the most efficient test to detect very small stroke related sensorimotor impairments. Time to contact and time to remove the stimulus separate out sensory and motor deficits, respectively [4]. We have modified the test to detect potential impairments of vibrissae sensation. Small adhesive tape square (2 × 2 mm) was placed on right or left C2 or C3 vibrissae of the mouse. The order of placement of the adhesive (right or left) was alternated between each animal and each session. The mouse was then placed in a home cage, and the times to contact and to remove each adhesive tape
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were measured, with a limit of 60 s. Four trials per testing day, two for each side for each animal were performed. Mice were tested before surgery and then 1, 7, 10 and 14 days after stroke induction.
(control group, Fig. 1A and C) and with intact subcortical structures were taken into further analysis. 3.2. Activity and exploratory behaviour
2.3.6. New object exploration Animals were tested for exploration of new objects in their home cages. All animals were presented with the same set of objects and none of the objects was presented twice within one session. Testing consisted of four 2-min. trials, with a 1-min. intertrial interval between each trial. Test was conducted before the stroke, 1, 4, 6, 10 and 18 days after the stroke. All procedures were conducted during the light cycle of the animal between 9 a.m. and 2 p.m. To control for possible odour cues the objects were cleaned with a 10% ethanol solution at the end of each trial. In each trial, the time to approach the object as well as the number of contacts and the total time of exploration of each object were measured using a stopwatch. Exploration was defined as direct contact of the whiskers, nose or front paws with the object. 2.3.7. Sensory labyrinth For sensory labyrinth test all the whiskers on one side of the snout were systematically trimmed (the other side remained intact) and mice were trained to pass through a labyrinth (30 × 40 cm) in the darkness using only tactile cues. Animals were rewarded with the food, which was placed always in the same location of the labyrinth. To increase motivation, food availability was restricted to 70% of daily rations during the whole period of the training and test. To control for possible odour cues, the labyrinth was cleaned with a 10% ethanol solution at the end of each trial. In the training phase each mice was placed in the labyrinth 4 times a day and this phase lasted until all animals were able to pass the labyrinth in 1 min. without returning to the start point. Than, stroke was induced within the barrel field contralateral to the intact whiskers. In the test phase each animal was tested twice at each time point: before the stroke, 1, 2, 7, 9, 11, 14, 15, 16, 17, and 18 days after ischemia. The average time of reaching the goal was then compared for sham operated and stroked animals.
There were no differences in the spontaneous motor activity between control (healthy and sham-operated pooled, n = 11) and stroke animals, regardless of the localization of the infarct in the barrel (n = 9) or visual cortex (n = 6), respectively, at the time of observation: 1 day (95.4% ± 1.07%, 93.57% ± 3.26%, and 89% ± 2.3%); 7 days (96.3% ± 1.6%, 93.73% ± 1.39% and 90.5% ± 5.5%) or 14 days (95.5% ± 1.8%, 97.28% ± 1.2% and 92.3% ± 7.85%) after stroke (ANOVA F(3,32) = 2.17, P = 0.1). Fig. 2A presents results of activity measurements for control and ischemic animals (group with the stroke within the barrel cortex). 3.3. Rotarod assessment of sensorimotor deficits Analysis of variance revealed no significant effect of time postlesion on rotarod performance (F(2,21) = 0.29, P = 0.75, Fig. 2B). The time spent on the rotarod by sham-operated (n = 6) and operated (stroke in the barrel cortex, n = 7) animals was comparable before and after the stroke: sham-operated—155.4 ± 18.44 s before the operation; 160 ± 10.32 s 1 day after and 166 ± 13.22 s 7 days after operation; operated animals: 168.4 ± 14.38 s before the stroke; 156 ± 9.23 s 1 day after and 168 ± 14.37 s 7 days after operation. There were no differences between mice with stroke in the barrel and visual cortex (data not shown). 3.4. Nest building test During analysis of the abilities of nest building, which is one of the species typical behaviour in mice, we failed to find a significant overall influence of stroke on nesting behaviour (Fig. 2C). There was no significant interaction between the infarct placement and paper towel nest construction (ANOVA P > 0.05, n = 24). 3.5. Whisker nuisance task (whisker response)
2.4. Verification of the infarct localization After completion of all tests animals were sacrificed, the brains were removed, frozen and cut on the cryostat. Representative sections from each brain were stained with cresyl violet to visualize the infarct localization and the longest axis of infarct was measured using a Nikon Eclipse 80i microscope equipped with a digital camera (Evolution VF; MediaCybernetics). 2.5. Statistical analysis The data were incorporated into a repeated measures ANOVA for parametric or Kruskal–Wallis for non-parametric analysis of variance. Specific effects were evaluated by Tukey or Dunn’s multiple comparison post-test, P < 0.05 was considered statistically significant. 3. Results 3.1. Size and localization of the infarct Lesions were about 2 mm in diameter (from 1.89 ± 0.6 to 2.33 ± 0.9 mm), and spanned all cortical layers leaving the subcortical structures intact. Only animals with verified size and localization of stroke embracing whole barrel cortex (experimental groups, Fig. 1A and B) or localized entirely beyond barrel cortex
A longitudinal evaluation of the uninjured sham animals (n = 8) and stroke animals irrespective of the stroke placement (n = 11) tested prior to, 1 day, week and 2 weeks after a sham injury did not show any aberrant behaviours in response to whisker stimulation (Fig. 2D). Whisker nuisance scores obtained by each particular group of tested animals were no higher than 11 at any time point, of a maximum 20 score. There were no significant differences between analyzed time points in injured animals (ANOVA, P > 0.05). Whisker Nuisance Task did not differentiate sham from ischemic mice over 2 weeks post-injury. 3.6. Adhesive removal test To remove the tape, animals raised both forelimbs towards their face and swiped off the stimulus with both forepaws. Typically, uninjured mice made contact and simultaneously removed the tape within 10 s. We did not observe significant differences between control (8.07 ± 0.65 s, n = 5) and sham operated animals (9.1 ± 0.98 s, n = 8) or stroke animals when adhesive tape was placed on whiskers ipsilateral to stroke (8.5 ± 0.63 s, n = 9). When stimulus was placed on whiskers contralateral to the infarct, one day after stroke the animals needed significantly more time to remove the tape than before the stroke (22.78 ± 3.4 s vs. 8.07 ± 0.65 s; ANOVA, F(4,44) = 15.54, P > 0.001, n = 9, Fig. 3D). However, there was no lag between contact and removing of stimulus. Seven days after stroke and at later post-stroke times there was no evidence of significant
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Fig. 1. (A) Schematic localization of photothrombotic infarct in the vicinity or within the barrel cortex, a cortical representation of sensory vibrissae and picture showing the infarcted brain. (B) Representative tangential sections stained with cresyl violet from control (left) and stroked (right) hemisphere showing the infarct within barrel cortex. (A–E) Rows of barrels. x-marking holes enabling alignment of consecutive slices. (C) Representative tangential and coronal sections stained with cresyl violet presenting localization of infarct within visual cortex beside the barrel field (BF) and showing that subcortical structures were intact.
extension of stimulus removal time in stroke animals (7 days poststroke: 6.78 ± 0.74 s; 10 days post-stroke: 7.94 ± 0.67 s and 14 days post-stroke: 8.67 ± 1.49; Fig. 3D). Initial contact with the stimulus went hand in hand with its removal which speaks for the absence of motor impairments. 3.7. New object exploration During the observation of stroked mice exploring a new object we noted significant effects of stroke localization on the animals’ behaviour. Mice with infarct localized in the barrel cortex spent significantly longer time exploring objects with vibrissae than control animals (17.67 ± 1.5 s vs. 6.22 ± 0.57 s) and that effect persisted for the whole period of post-stroke observation (ANOVA, F(5,53) = 2.45, P = 0.001, n = 9) (Fig. 3A). Detailed results for particular post-stroke days are summarized in Table 1A. In this group of animals we did not observed any significant interaction between stroke and the time the animals needed to approach the object (ANOVA, F(5,53) = 0.5, P > 0.05) or in the number of contacts with the objects (ANOVA, F(5,53) = 2.57, P > 0.05) (Table 1A). In the group of mice with infarct outside of the barrel cortex, embracing part of visual cortex, a significant interaction between the stroke and the time of approaching the object could be observed (ANOVA, F(5,25) = 6.4, P > 0.02, n = 6). Animals with stroke needed more time to approach the object (10 ± 2.72 s before the stroke vs. 20.13 ± 3.6 s 1 day after stroke). The observed effect was transient and after one week of recovery was no longer observed (16.75 ± 9.5 s) (Table 1B, Fig. 3B). This group of mice did not show any effect of stroke on the time spent on the object exploration or on the number of contacts with objects (ANOVA, F(5,33) = 2.04, P > 0.05, Table 1B). 3.8. Sensory labyrinth We have observed a considerable influence of stroke upon performance in the labyrinth. Mice with stroke in the vibrissae representation were unable to complete the task, which they had learned before the stroke. Before the stroke the passage through
labyrinth took them in average19 ± 3.8 s whereas one day after the stroke they needed 64.3 ± 26.6 s in average, which was more than three times longer (ANOVA, F(10,66) = 13.41, P = 0.02, n = 8) (Fig. 3C). For sham-operated animals the passage time was similar for the whole tested period and amounted 13.9 ± 3 s (Fig. 3C, n = 6). After 14 days of recovery stroke animals re-learned to navigate straightforward to the finish point in the labyrinth with average time of 13 ± 1.5 s. Detailed results for consecutive days are presented in the table (Table 2).
4. Discussion We assessed sensorimotor functions of mice after the focal photothrombotic stroke. We found that unilateral photothrombotic injury within or in the neighbourhood of barrel cortex, a part of primary somatosensory cortex, produce restricted, modality specific behavioural consequences in the acute post-stroke period. Our earlier data have shown that in rats and mice, vicinity of infarct impairs functional cortical plasticity induced by sensory deprivation of vibrissae. [14,18,19]. Here, using a battery of behavioural tests (global and modality specific) we evaluated the sensory functions in the acute post-ischemic phase, to establish which functions might be the most susceptible to the detrimental effects of stroke. Earlier, we and others showed that ischemic injury of the whisker representation impairs performance of gap-crossing task and this effect is still present more than one month after stroke [7,20,21]. We did not observed any differences in the normal exploratory behaviour and activity between healthy and operated animals even as early as one day after surgery. That implied that animals did not suffer from post-surgery pain or depression that could interfere the performance of behavioural test we used. Accelerated rotarod was used to assess the motor coordination and balance alterations. Earlier publications reported the impairment of rotarod performance after photothrombotic stroke, but the infarct embraced limbs representation in the sensorimotor cortex [22,23]. Here, the infarct was localized in the proximity of forepaw representation, but it did not interfere in its functioning, since
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Fig. 2. (A) Chart showing results of activity and exploratory behaviour analysis. Data are presented as a percent of pre-ischaemic activity. No differences were observed during two weeks after stroke between sham-operated and stroke groups. Data represent mean ± SEM. (B) Sensorimotor coordination and balance remained unaffected during the first post-stroke week. Chart presents the time during which animals were able to stay on the rod (s). Data represent mean ± SEM. (C) Nesting behaviour was not significantly impaired after stroke. There was no difference between sham-operated and stroke animals. Chart present points average points given by two unrelated observers. Data represent mean ± SEM. (D) Results of whisker nuisance test showing no significant differences in whiskers reactivity to the tactile stimulus between sham-operated and stroke animals during two post-stroke weeks. Chart presents an average points given by two unrelated observers. Data represent mean ± SEM.
ischaemic animals did not differ from the control group according to the rotarod performance. We have assessed also the nesting behaviour, which is one of the maintenance related behaviours, displayed by both sexes in parental and non-parental contexts and is controlled by levels of arousal and motivation [24]. Nest building was impaired by medial prefrontal and hippocampal lesions in rats and hamsters but not in mice [15,25–27] suggesting that this result might be species specific. Our mice presented normal nesting activity implying proper functioning of hippocampus and prefrontal cortical areas.
To estimate the vibrissae-mediated perception we have conducted several somatosensory specific tests. Whisker Nuisance test allows to assess the reactivity to the tactile stimulus and was effective in detecting post-traumatic morbidity following the diffuse brain injury in rats. After brain injury animals were overactive to the tactile whisker stimulation what was related to the stress response [28–30]. In our experiments we did not detect any change in animals’ reactivity to the whisker-delivered stimulus after ischemia irrespective of the infarct localization. On the other hand, in the adhesive removal test early after stroke we have observed
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Fig. 3. (A) Chart showing results of new objects exploration test in animals with infarct within barrel cortex. After stroke animals spent significantly more time exploring new objects than before ischaemia. The effect sustained throughout whole observation period (18 days after stroke). Data represent mean ± SEM. * P < 0.05; ** P < 0.01. (B) Chart showing results of new objects exploration test in animals with infarct within visual cortex. One day after stroke animals needed significantly more time to achieve object than before ischaemia. This effect could not be observed a week after stroke. Data represent mean ± SEM. * P < 0.05. (C) Results of sensory labyrinth test showing the impairment of labyrinth performance one day after stroke and recovery of function after 15th post-stroke day. Sham-operated animals were unaffected. Data represent mean ± SEM. ** P < 0.01. (D) Results of adhesive removal test. Chart represents the time needed to contact and remove the stimulus from whiskers contralateral to the injured barrel cortex. Sensory deficits could be observed one day after stroke with the recovery within the first post-stroke week. Data represent mean ± SEM. *** P < 0.001. Table 1 Results for novel object exploration task for animals with stroke within the barrel cortex (A) and beside the barrel cortex within the area of visual cortex (B). (A). Parameter/time (s); average ± SEM
Before stroke
1 day
4 days
6 days
10 days
18 days
Time to approach Number of contacts Time of exploration
32.44 ± 5.3 3 ± 0.52 6.22 ± 0.57
23.44 ± 11.58 5.22 ± 0.74 17.67 ± 1.5*
33.11 ± 9.19 4 ± 0.66 20.67 ± 3.4**
43.22 ± 7.94 3.4 ± 0.55 23.11 ± 6.6**
31.11 ± 6.75 5.3 ± 0.62 25.89 ± 6.5*
34.44 ± 11.29 6 ± 1.15 19.78 ± 3.1*
(B) Parameter/time (s); average ± SEM Time to approach Number of contacts Time of exploration * * **
Before stroke 10 ± 1.7 3.5 ± 0.42 8.3 ± 2.48
1 day 21.3 ± 2.7* 4.2 ± 0.7 9.3 ± 2.4
Significant difference with the result before the ischemia; 1 day, 4 days, etc.—days after stroke. Data represent mean ± SEM. P < 0.05. P < 0.01.
7 days 16.83 ± 3.06 4.1 ± 0.4 11.3 ± 2.3
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Table 2 Time to complete the sensory labyrinth task for sham-operated and ischemic animals.
*
Group/time (s); average ± SEM
Before stroke
1 day
2 days
7 days
9 days
11 days
14 days
15 days
16 days
17 days
18 days
Sham operated Ishemic
13.5 ± 1.1 19.3 ± 2.4
14.0 ± 1.0 64.3 ± 6.9**
15.5 ± 1.5 66.3 ± 5.7**
12.5 ± 0.9 73.2 ± 11.1***
11.5 ± 1.6 57.2 ± 7.0**
15.0 ± 1.4 52.0 ± 2.4**
14.0 ± 1.1 67.2 ± 5.8***
13.0 ± 1.7 9.2 ± 0.6
18.0 ± 1.7 21.3 ± 2.3
11.0 ± 1.2 14.8 ± 1.1
14.5 ± 1.4 13.0 ± 1.1
Significant difference with the result before the ischemia; 1 day, 2 days, etc.—days after stroke. Data represent mean ± SEM. ** P < 0.01. *** P < 0.001.
significant stroke-related impairments illustrating the infarctinduced sensory deficit. In the whisker nuisance test however, tactile stimulation is much stronger and spared subcortical vibrissal centres may be sufficient for the stimulus processing. Correlations between cortical lesions and ischemia-induced behavioural impairments in the adhesive removal test have been well investigated in the literature and many authors have demonstrated a close correlation between contralateral removal latencies on this task and changes in the forelimb cortex following transient MCAO and photothrombosis [31–34]. In those experiments however, observed impairments lasted at least 3 weeks, whereas in our investigations sensory deficits were visible only early after stroke with recovery within the first post-stroke week. This suggests rapid activation of compensatory mechanism aiming at retaining of the basic tactile sensation. Novel object exploration examined the reactivity of ischemic animals to novelty, and the basic premise of this test is that animals have the natural curiosity which impels them to explore novel environments. The aim of the test was to estimate if unilateral ischemic disruption of barrel cortex will influence the exploratory behaviour of mice. Our data indicate that animals with the stroke within barrel cortex explored objects for a significantly longer time than the control group. The time of object approaching and the number of contacts with objects did not change which suggests unaffected reactivity of injured animals. Thus, we suggest that mice needed more time to collect all necessary information about each object. The observed effect persisted through the entire observation period and we did not observed behavioural recovery. Although, based on the Whisker nuisance Test results it may seem that touch sensation is not impaired after ischemic injury of barrel cortex, we need to note that here, animals need to collect more complex information about size, structure and texture of the object and integrate them in the higher brain areas. Also, those two task differ in nature, with Nuisance test relying on involuntary (passive) and Novelty test on voluntary (active) touch perception. The novel object exploration test turned to be sensitive to the stroke localization. Mice with unilateral infarct beside the barrel field but embracing significant part of visual cortex needed more time to approach the object while the number of contacts and time of exploration remained unchanged. Since this effect was transient and could be observed only one day after stroke, we suggest that it was caused by some disturbances of visual symmetry, which could be easily overcome when animals accommodate to changed visual perception. Cognitive ability was checked with the sensory labyrinth test. It involves spatial and working memory assessment and tactile navigation through the labyrinth in the dark requires sensory information from the whiskers similarly like during subterranean burrows behaviour [35,36]. Our labyrinth task was conducted in the dark since to avoid usage of visual cues [37,38]. Stroke within the representation of intact whiskers resulted in initial inability to solve the labyrinth. However, we observed the complete recovery after 14 days. Times that animals needed to learn and than re-learn the labyrinth task after stroke were comparable what suggests that memory about this task performance was located in
the destroyed cortex and acquisition of memory had to start from the beginning. Earlier studies that have examined the behavioural results of barrel cortex infarction presented diverse effects on the cognitive tests performance, but above all on the recovery and return of function. Guic-Robles and colleagues have shown that roughness discrimination task is barrel cortex-dependent, and after its bilateral lesion no behavioural recovery can be observed [39]. In other study Hurwitz et al. [40], have tested animals in the Gap Crossing Test and showed robust behavioural deficits, but with 80% recovery. Then, Pazos et al. [41] have demonstrated that behavioural recovery of rats after photothrombosis tested in T-maze is not dependent upon the remaining somatosensory cortical tissue. Moreover, they suggested that neither the contralateral somatosensory cortex nor the vibrissal representation within ipsilateral secondary somatosensory cortex play a critical role in the observed recovery process. Our studies does not allow us to take a stand on this dispute, however, since in our investigations both, contralateral primary somatosensory cortex and secondary somatosensory cortices remained intact, they could possibly take part in restoration of behavioural functions after ischemia. Barrel neurons are influenced also by the whiskers on the ipsilateral side of the face and this response is mediated via callosal connections [42,43], which are supposed to provide at least part of the pathway for signaling the presence of an infarct. Behavioural studies have shown that tactile learning occurring in one barrel cortex leads to significant transfer to the homologous barrel on the other side [44]. Also, secondary somatosensory cortex (SII), which contains the representation of facial vibrissae and is reciprocally connected with the ipsilateral primary somatosensory cortex [45,46] could possibly support the post-stroke functional recovery. It is postulated that SII receives direct input from the thalamus independently from SI [47] and we have shown that the response of SII cortex to vibrissal input was modified bilaterally after classical conditioning which involved unilateral tactile stimulation [48]. Thus, after unilateral injury of primary barrel cortex, information acquired with the involvement of uninjured subcortical structures might be directed towards the spared areas of ipsilateral cortex or to the contralateral homotopic cortex.
5. Conclusions Photothrombotic stroke within cortical representation of sensory vibrissae in its acute phase induced sensory deficits detectable with specific behavioural tests. Basic sensation of whisker stimulation was spared with strong stimulus (whisker nuisance test) while impaired when weaker tactile stimuli (adhesive removal test) were applied. Cognitive sensory abilities were impaired as revealed by sensory labyrinth task performance, however animals retained abilities of task re-learning. Pronounced and long-lasting sensory deficits were visible when animals was confronted with new environmental cues that had to be explored with vibrissae (new object exploration test). For this test no functional recovery could be observed.
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