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FGF-2 induces behavioral recovery after early adolescent injury to the motor cortex of rats
Behavioural Brain Research 225 (2011) 184–191
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Research report
FGF-2 induces behavioral recovery after early adolescent injury to the motor cortex of rats Farshad Nemati ∗ , Bryan Kolb Department of Neuroscience, University of Lethbridge, Lethbridge, AB, Canada, T1K 3M4
a r t i c l e
i n f o
Article history: Received 21 April 2011 Received in revised form 9 June 2011 Accepted 13 July 2011 Available online 23 July 2011 Keywords: Adolescence Motor cortex injury Basic fibroblast growth factor Dendritic reorganization Functional recovery
a b s t r a c t Motor cortex injuries in adulthood lead to poor performance in behavioral tasks sensitive to limb movements in the rat. We have shown previously that motor cortex injury on day 10 or day 55 allow significant spontaneous recovery but not injury in early adolescence (postnatal day 35 “P35”). Previous studies have indicated that injection of basic fibroblast growth factor (FGF-2) enhances behavioral recovery after neonatal cortical injury but such effect has not been studied following motor cortex lesions in early adolescence. The present study undertook to investigate the possibility of such behavioral recovery. Rats with unilateral motor cortex lesions were assigned to two groups in which they received FGF-2 or bovine serum albumin (BSA) and were tested in a number of behavioral tests (postural asymmetry, skilled reaching, sunflower seed manipulation, forepaw inhibition in swimming). Golgi–Cox analysis was used to examine the dendritic structure of pyramidal cells in the animals’ parietal (layer III) and forelimb (layer V) area of the cortex. The results indicated that rats injected with FGF-2 (but not BSA) showed significant behavioral recovery that was associated with increased dendritic length and spine density. The present study suggests a role for FGF-2 in the recovery of function following injury during early adolescence. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Basic fibroblast growth factor (FGF-2) plays a role in the regulation of cerebral development and in modulating recovery from cerebral injury [e.g., 3,6,18,20,21,27]. The effects of FGF-2 on functional recovery are especially large in animals with cortical injury in infancy, but the mechanisms appear to vary with age. Thus, animals with medial prefrontal lesions on postnatal day 3 show enhanced recovery that is associated with increased cortical thickness and dendritic arborization [e.g., 3]. In contrast, animals with motor cortex lesions on postnatal day 10 show enhanced motor recovery that is associated with the regeneration of the lost tissue [20–22]. This regeneration does not occur if the injuries are on day 3 or in adulthood or if the day 10 lesions are in more posterior cortex. The goal of the current study was to determine if FGF-2 would enhance recovery from motor cortex injuries in early adolescence either by stimulating the proliferation of new cells or the hypertrophy of cortical pyramidal cells. We have previously shown that rats given unilateral motor cortex lesions in early adolescence (postnatal day 35, P35) showed significant deficits on various motor tasks and no change in dendritic organization in perilesional or contralateral motor cortex
∗ Corresponding author. Tel.: +1 403 329 2044; fax: +1 403 329 2775. E-mail address: [email protected] (F. Nemati). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.07.023
pyramidal cells [23]. This result contrasted to the effects of late adolescent (P55) motor cortex lesions whereby animals showed nearly complete recovery associated with extensive dendritic hypertrophy. We therefore knew that adolescent rats were capable of significant functional recovery but that this did not happen if the injuries were in early adolescence. We reasoned that if early adolescent rats were capable of similar functional recovery, then it could be stimulated by postinjury application of FGF-2. The results confirmed our prediction. 2. Material and methods 2.1. Animals Female Long–Evans hooded rats were raised in the University of Lethbridge vivarium and group-housed in two or three individuals in different cages with bedding in the colony room. The temperature of the colony room maintained at 22 ◦ C on a 12 h light/dark cycle. The animals were assigned either to experimental or control groups in which they received basic fibroblast growth factor “FGF-2” (n = 5) or bovine serum albumin “BSA” (n = 6) respectively for seven consecutive days starting the day after placing motor cortex lesion in their left hemisphere. The University of Lethbridge Animal Care Committee approved experimental procedure in this study on behalf of the Canadian Council of Animal Care. We chose to use females in this study for two reasons. First, we used females in our previous study looking at P35 and P55 rats. Second, we elected to look at females because we had previously found that although FGF-2 did not affect most measures of recovery in a sexually-dimorphic manner, there was a differential effect on body weight [3]. Specifically, FGF-2 reduced body weight in both sham and lesion males but had no effect in females. Given that we were looking at motor behaviors and given that differences in body size can affect performance in motor tasks (and
F. Nemati, B. Kolb / Behavioural Brain Research 225 (2011) 184–191 especially the skilled reaching task because the bars are relatively further apart for smaller animals) we wanted to control for body weight differences. 2.2. Surgical procedures Unilateral motor cortex lesions were placed in the left hemisphere of the postnatal 35 (P35) day old rats. A standard procedure was followed to generate the lesions in which each animal was anesthetized in an induction chamber using 4% isoflurane first. Then rats were positioned in a stereotaxic device that was modified to fit the size of their body. During the surgery the animals were kept anesthetized with 2–3% isoflurane and the aspiration method was used to produce the lesions. In order to remove the motor cortex tissue an area of the skull corresponding to the anterior motor cortex was removed. The coordinates were from AP +2 to −2 and lateral from +1.5 to the temporal ridge. Following the removal of the dura, the tissue of motor neocortex was gently sucked out using a fine pipette. As soon as the lesion was completed and rats reached homeostasis the incision was sutured using the sterile 5-0 Vicryl suture. 2.3. FGF-2 injections FGF-2 diluted in BSA (0.1 M PO4 as a concentration of 1 mg/ml) at the concentration (1 g/ml) was injected s.c. at 0.1 ml/10 g of body weight. The concentration was demonstrated to be efficient in enhancing neurogenesis in P7–P28 in Wagner et al. [33] study, which was closest age to our sample (P35). Also, it has been shown that this concentration contributes to the recovery after motor cortex injury in developing rats [20].
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a corner of a Plexiglas box (45 cm × 14 cm × 35 cm) during each training or testing session. Animals were trained for four consecutive days and their behavior was video recorded on the last day. Two measures were used to quantify the behavior of animals: (1) time: the total amount of time spent on manipulating, opening the shell and consuming the seeds and (2) the number of pieces of shells left after animals retrieve and eat the seeds. If the pieces were too small to count, a maximum of 30 pieces of shells was assigned.
2.4.4. Forepaw inhibition swimming task Being semi-aquatic animals, rats only use their hind limbs for propulsion, while keeping their forelimbs immobile under their chin while swimming [19]. Motor cortex lesion is usually associated with the disinhibition of the forelimb (strokes) during the swimming in rats [17]. To evaluate the effect of FGF-2 treatment following motor cortex lesion on the swimming of the rats in this task the animals were trained for 4 days in a rectangular aquarium (120 cm × 43 cm × 50 cm). Rats had to swim from one end of the aquarium to a visible platform placed at the other end of the rectangular. Animals were released in the water (25 ◦ C) in one side of the rectangular to swim towards the platform on the other side on the training days. Each rat was given several consecutive trials (≈10 trials) to learn to swim strait without touching the wall of the pool. On the fourth day, animals’ behavior was video recorded while swimming to the platform. The dependent variable was the number of strokes during swimming to the platform without touching the wall.
2.5. Anatomical procedures 2.4. Behavioral procedures The behavioral tests (cylinder, tray reaching, sunflower seed, forepaw inhibition) began 1 month after surgery. In order to motivate the animals in the reaching and sunflower seed tests, rats were placed on a light food-restriction regime in which each rat obtained 20 g of food per day. Rats were fed 30 min after testing session every day. The body weight of the rats was monitored every day and maintained at ≈95–98% during the behavioral testing. 2.4.1. Cylinder test In order to measure the use of forelimb for weight support rats were tested in the cylinder test in which rats explore their environment vertically [28,29]. Each rat was placed in a transparent cylinder 20 cm in diameter and 30 cm high. The dependent variable was the number of contacts by each forepaw with the cylinder wall while simultaneous contact of limb with the wall of the cylinder was counted as a touch for each paw. Rats were placed gently in the cylinder to explore it for four consecutive days each day for 3 min. The animals’ explorative behavior inside the cylinder was videotaped on the last day. Using a mirror at an appropriate angle underneath the cylinder the experimenter was able to videotape the animals’ behavior from a ventral view. 2.4.2. Tray reaching task The forelimb movement skills were assessed in Tray reaching boxes that are designed to measure the natural behavior of food consumption in rats [19]. Each box was surrounded by clear Plexiglas on three sides and thin metal bars on the fourth side in the front. A food tray (4 cm wide and 0.5 cm deep) was positioned in front of the bars. In order to prevent the rats from dragging the chicken feed pellets into the box to consume them there was a small gap between the tray and testing box. The bottom of the test box was wired in a way that food pellet could fall through which and therefore rats could not pick up the food from the floor. In order to perform the task successfully rats had to reach and grasp the small pieces of food after passing the bars installed in front of the box, and skillfully retract their paws and consume the food. One month after surgery rats with motor cortex lesion in both BSA-injected and FGF2-injected group were trained in Tray reaching task for 10 consecutive days. The behavior of rats in the tray-reaching box was videotaped on the last day. The success percent was calculated according to the following formula:
success percent =
hit × 100 reach
Success percent was defined in terms of two variables: (1) hit defines a reaching movement in which a rat successfully inserts its paw through the bars, grasp the food pallet on the tray and eat the food in the box following retracting its paw; (2) reach includes both successful movements [hits] and unsuccessful movements [attempts]. 2.4.3. Sunflower seed task Rats use their limbs and digits during sunflower seed consumption [34]. In order to consume a seed rat first starts manipulating the seed and position it into an appropriate angle in relation to their mouth before shelling them. After rats chew a piece of shell away then they can split the seed into two halves longitudinally with a final bite [34]. In order to evaluate the animals’ ability to open and successfully consume seeds using their limbs and digits, five sunflower seeds were placed in
An overdose of sodium pentobarbital was given intracardially to each animal, which was perfused using 0.9% saline at the conclusion of behavioral tests. The brains of the animals were removed from the skull and immersed in 20 ml of Golgi–Cox solution. Following 14 days in Golgi–Cox solution the brains were transferred to 30% sucrose solution for 7 more days. The brains then were cut on a vibratome at 200 m [7], and the sections were mounted on 2% gelatin slides. The slides were stored in a humidity chamber until staining. Immediately after staining the brains were coverslipped using Permount.
2.5.1. Dendritic analysis The dendritic processes had to meet the following inclusive criteria to be selected as a sample for drawing procedure: (1) the dendritic processes should not be affected by stain precipitations, blood vessels, or heavy clusters of dendrites from other cells; (2) the apical and basilar dendrites must be visible on the section. The morphology of neurons was studied in three areas of neocortex: layer III of parietal area ipsilateral or contralateral to the lesion side and also layer V of forelimb area contralateral to the lesion side [35]. After drawing cells in the abovementioned area using the camera lucida the following measures were used in analyzing the structure of cells: total dendritic length, branch numbers, and spine density. In order to determine the branch numbers and total dendritic length, the neurons were drawn under camera lucida set for the magnification of 200×. Branches originating from the cell body were considered as basilar dendrites, and those originating from the main dendrite projected from the cell body as apical dendrites. The total length of dendrites was measured using Sholl’s [30] concentric ring procedure [31]. This method includes both the principal apical dendrite as well as all branches from it. The mean of total dendritic length (in m) was estimated through multiplying the mean total number of intersections by 20 m, which is the interval between concentric spheres from the center of the cell body. The numbers of spines on branch order of 2 or 3 of apical and that of 4 or 5 of basilar dendrites were counted using a camera lucida set for the magnification of 1000×. The density of spines is presented as the number of spines/10 m. We chose to make the spine measurements on these branch orders because we had seen effects of the injuries in these regions in our previous study.
2.5.2. Cortical thickness Golgi–Cox stained sections were projected on a Zeiss 2 POL projector set at a magnification of 13×. The thickness of motor neocortex was measured at medial, central and lateral points in the contralateral cortex at 5 Paxinos and Watson [24] planes: plane 10 (areas M1, S1J, AID); plane 18 (areas M1, S1BF, G1); plane 25 (areas M1, S1BF, G1); plane 37 (areas V2m, AuD, TeA); and, plane 43 (areas V2MM, V1B, TeA). Similar measurements were made at the central or lateral points on the lesion side. See Fig. 1 in Stewart and Kolb [31] for an illustration.
2.6. Statistics Repeated measure analysis of variance (ANOVA), and also unpaired t-test were used to analyze the data. Bonferroni t-test was used for post hoc analysis. Also, Pearson correlation was used to study the relationship between variables. Significant values have been illustrated by “*” to indicate p < 0.05 and by “**” to indicate p < 0.01.
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Fig. 1. Cylinder task. Rats in FGF-injected group made fewer numbers of contacts with the wall of the cylinder than rats in BSA-injected group. **p < 0.01.
3. Results 3.1. Behavioral analysis The behavior of rats assigned to FGF-2 or BSA groups was compared in four different motor behavior tasks (cylinder task, tray reaching test, sunflower seed task, forepaw inhibition swimming task) sensitive to limb and/or digit movements. In general, rats that received FGF-2 following unilateral motor cortex lesion in their left hemisphere at 35 days of age performed better in behavioral tasks as compared with those receiving BSA following the lesion. 3.1.1. Cylinder task While exploring the cylinder in a vertical position rats used their forelimb to support their weight against the cylinder wall. Rats in BSA group with motor cortex lesion generated at P35 touched the cylinder wall more frequently in comparison with rats in FGF-2 group with the same lesion. In general, the left forelimb (ipsilateral to the lesion side) was used more frequently to support animals’ weight against the wall than right forelimb (contralateral to the lesion side) regardless of animals’ group. Statistical analysis of the data confirms the observations. The following represents the formal analysis of data as illustrated in Fig. 1. The time spent on vertical exploration of the cylinder was not significantly different between groups (t[9] = 0.6, p = 0.55). Repeated measure ANOVA revealed a significant effect of group (F[1,9] = 11.51; p < 0.009) in the number of touches with the wall of the cylinder and a significant effect of limb use (F[1,9] = 6.08; p < 0.04) during the vertical exploration in which the limb ipsilateral to the lesion side was used more frequently. Also, there was no significant interaction of limb use by group (F[1,9] = 3.47; p = 0.09).
Fig. 2. Tray reaching task. Rats with motor cortex injuries that received FGF-2 were more successful in the reaching task than those in BSA-injected group. *p < 0.05.
injected group were more skillful than those in BSA-injected group and thus spent less time retrieving and consuming the seeds. Nevertheless, the number of pieces of the shells left after retrieval of the seeds was not significantly different between groups. Fig. 3 illustrates the performance of rats in the task. Unpaired t-test indicated a significant difference between groups (t[9] = 2.41, p < 0.04) in terms of time spent on retrieving and consuming seeds. The number of shells left after retrieval was not significantly different between groups (t[9] = 0.16, p = 0.87). 3.1.4. Forepaw inhibition swimming task Although one rat did not learn to swim towards the platform without touching the wall, others were matched in terms of the
3.1.2. Tray reaching test After 10 days of training in Tray reaching task, one rat in each group did not reach for food at all and was eliminated from the analysis. Two rats in the FGF-injected group reached for food with their ipsilateral paw while two other rats were ambidextrous. In BSA group one rat was ambidextrous and other rats used their ipsilateral paw when reaching. Rats with unilateral motor cortex lesion at P35 demonstrated more successful reaching behavior following FGF-2 injections in comparison with rats that received their lesion at P35 but were injected BSA as shown in Fig. 2. Unpaired t-test indicated a significant difference between groups (t[7] = 2.38, p < 0.05). 3.1.3. Sunflower seed task Following 3 days of training in sunflower seed task, the behavior of rats were video recorded on the following day. Rats in FGF2-
Fig. 3. Sunflower seed task. (A) Rats in both FGF-2 and BSA-injected groups demonstrated almost the same level of skill in shelling the seeds measured in terms of the pieces of shells left. (B) Rats in FGF-2 group spent shorter period of time shelling and consuming the seeds than those in the BSA-injected group. *p < 0.05.
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no significant effect of forelimb use in terms of the number of strokes (F[1,8] = 1.86; p = 0.2), and no significant interaction of forelimb use by group (F[1,8] = 2.99; p = 0.12).
3.2. Histological results The lesions included the entire forelimb region of the left cerebral cortex as well as much of the body and some of the hindlimb region as illustrated in Fig. 5. No direct injury to the underlying striatum was observed.
3.2.1. Brain weight Unpaired t-test indicated no significant difference (t[9] = 1.93, p = 0.08) between groups in terms of the brain weight. Fig. 4. Forepaw inhibition task. Rats in FGF-2 group made fewer strokes during the swimming than those in BSA group. **p < 0.01.
number of trials that they were able to swim towards the platform without touching the wall of the aquarium during the videotaping. Rats in FGF2-injected group showed dramatic improvement in their swimming towards the platform as compared with the rats in BSA-injected group as shown in Fig. 4. Repeated measure ANOVA indicated a significant effect of group (F[1,8] = 11.47; p < 0.01), but
3.2.2. Cortical thickness The thickness of cortex was measured in the medial, central, and lateral points in the hemisphere contralateral to the lesion. Because the lesion had expanded towards the midline suture in the lesion side the thickness of the cortex was measured only at the central and lateral points in the lesion hemisphere. There was no group effect of cortical thickness in either hemisphere.
Fig. 5. Serial drawings of coronal sections of Golgi–Cox-stained tissues of the brains with motor cortex lesions. Brains from each group were ranked by lesion size and the median lesions were chosen as representative examples.
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Fig. 6. Cortical thickness at the medial point contralateral to the lesion hemisphere. The histobars from right to left in each group (BSA or FGF) corresponds to the plane 10 (areas M1, S1J, AID); plane 18 (areas M1, S1BF, G1); plane 25 (areas M1, S1BF, G1); plane 37 (areas V2m, AuD, TeA); and, plane 43 (areas V2MM, V1B, TeA). A dramatic increase in the thickness of the cortex from plane 18 (areas M1, S1BF, G1) to plane 25 (areas M1, S1BF, G1) was observed in FGF-2 injected rats compared to those in BSA-injected group. *p < 0.05.
3.2.3. Ipsilateral cortical thickness ANOVA indicated that the measures of cortical thickness in the central sections ipsilateral to the lesion are not significantly different (F[1,9] = 0.11; p = 0.74) between groups. There was a significant effect of planes (F[4,36] = 10.12; p < 0.0002), but no significant interaction of group by planes (F[4,36] = 0.49; p = 0.73). The plane effect reflects the normal thinning of the cortex in a rostral–caudal direction. The group effect was not significant (F[1,9] = 0.13; p = 0.72) in the lateral points, but a significant effect of thickness (F[1,9] = 18.60; p < 0.0002) was found in planes of the central points representing the normal thinning going from anterior to posterior cortex. There was no interaction of plane by group (F[4,36] = 2.43; p = 0.06). 3.2.4. Contralateral cortical thickness ANOVA indicated no significant difference between groups in cortical thickness of medial section contralateral to the lesion side (F[1,9] = 0.002; p = 0.96). There was a significant difference between the cortical thickness in different planes (F[4,36] = 39.10; p < 0.0002) that were in interactions with the group (F[4,36] = 2.75; p < 0.05). Post hoc analysis indicated a significant increase in the thickness of the cortex from plane 18 (areas M1, S1BF, G1) to plane 25 (areas M1, S1BF, G1) in FGF-2 injected rats compared to those in BSA-injected group (Fig. 6). ANOVA also indicated no significant effect of group in the measures of cortical thickness in the central section of contralateral hemisphere (F[1,9] = 1.50; p = 0.25). There was a significant effect of planes (F[4,36] = 21.83; p < 0.0002) that were in no interaction with the group (F[4,36] = 1.07; p = 0.38). No significant difference was found between groups in cortical thickness of the lateral section (F[1,9] = 0.02; p = 0.89). There was a significant difference between the cortical thickness of different planes (F[4,36] = 27.88; p < 0.0002), with no significant interaction by group (F[4,36] = 2.16, p = 0.09). 3.2.5. Morphological analysis of neurons in parietal cortex (layer III) 3.2.5.1. Dendritic length (Sholl analysis). The Golgi analysis demonstrated that the total length of basilar dendrites in FGF-2 group was longer than those that received BSA injection after receiving the same lesion at the same age (P35). The total length of apical dendrite of neurons was not significantly different between groups.
Fig. 7. Dendritic length of neurons (in m) in parietal cortex (III). The total length of dendrites in apical field (A) was virtually the same in both FGF-2 and BSA-injected groups, but in the basilar field (B) it was greater in rats that received FGF-2 injections than those in BSA-injected group. *p < 0.05.
The following section represents the formal analysis of the results illustrated in Fig. 7. A two way ANOVA revealed a significant effect of group (F[1,9] = 6.24; p < 0.04) in dendritic length of basilar dendrites of pyramidal neurons in layer III of parietal cortex. There was no significant effect of hemispheres (F[1,9] = 1.01; p = 0.34) nor an interaction of hemispheres by group (F[1,9] = 0.43; p = 0.52). In contrast, ANOVA on apical dendrite found no significant effect of group (F[1,9] = 1.12; p = 0.31) hemisphere (F[1,9] = 0.10; p = 0.75), or the interaction (F[1,9] = 0.04; p < 0.84) in the length of apical dendrites.
3.2.5.2. Branch order. There were no differences in the number of basilar or apical dendritic branches in parietal cortex (layer III). A two way ANOVA (treatment × hemisphere) indicated no significant effect of either treatment in branch number of basilar dendrites (F[1,9] = 2.84; p = 0.12) hemisphere (F[1,9] = 0.46; p = 0.51) or the interaction of hemisphere by group (F[1,9] = 0.79; p = 0.39). Similarly, ANOVA found no significant effect of treatment, hemisphere or the interaction for the apical dendrites (F[1,9] = 0.42; p = 0.53, F[1,9] = 0.26; p = 0.61; F[1,9] = 0.09; p = 0.76).
3.2.5.3. Spine density. There was an increase in spine density of the basilar, but not apical, dendrites in the ipsilateral, but not in the contralateral hemisphere, in the FGF-2 group relative to the BSA group (Fig. 8). A two-way ANOVA revealed no group effect in spine density in the basilar dendrite (F[1,9] = 4.21; p = 0.07) but there was a significant effect of hemisphere (F[1,9] = 10.26; p < 0.02) and the group × hemisphere interaction (F[1,9] = 9.41; p < 0.02). In contrast, there were no significant effects of group, hemisphere, or the interaction for the apical dendrites: (F[1,9] = 0.97; p = 0.35, F[1,9] = 0.24; p = 0.63, F[1,9] = 2.79; p = 0.12).
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4. Discussion The two primary findings of the current experiment were: (1) there is continuing evidence for age-dependent patterns of functional recovery after cortical injury during development; and (2) the administration of FGF-2 alters cerebral function and morphology after P35 motor cortex lesions. We consider each finding separately. 4.1. There are age-dependent effects of cortical injury during development
Fig. 8. Spine density (per 10 m) in parietal cortex (III). The density of spines in basilar field of parietal cortex ipsilateral (I) but not contralateral (C) to the lesion side was greater in FGF-2 group than those in BSA-injected group. *p < 0.05.
3.2.6. Morphological analysis of basilar fields of neurons in contralateral forelimb area of cortex (layer V) 3.2.6.1. Dendritic length. The total length of dendrites was longer in rats with motor cortex lesion treated with FGF-2 than those in BSAinjected group (M’s = 1709.6 ± 210.33; M’s = 1214.66 ± 110.55). Statistical analysis of data using unpaired t-test indicated a marginally significant difference between groups (t[9] = 2.19; p < 0.06) in dendritic length of basilar field in layer V neurons of forelimb area of the cortex. 3.2.6.2. Branch order. The number of branches in contralateral hemisphere of the brain was almost the same in both groups (M’s = 28.96 ± 3.63; M’s = 23.03 ± 1.93). Unpaired t-test found no significant effect of group in branch order (t[9] = 1.51; p = 0.16). 3.2.6.3. Spine density. Although there was a trend for an increase in spine density in the FGF-2 compared to BSA group (M’s = 9.54 ± 0.64; M’s = 8.15 ± 0.35), the effect was not significant (t[9] = 1.97; p = 0.08). 3.3. Correlations Pearson correlation was used to study the relationship between the behavioral measures and reorganized dendritic measure (dendritic length) of forelimb cortex (V) as an area responsible for direct control of forepaw movements. The analyses indicated a meaningful relationship between dendritic length in the forelimb area contralateral to the lesion side and motor behaviors. The analysis revealed a significant correlation (r = 0.67; p < 0.05) between dendritic length in the forelimb area contralateral to the lesion side and success in tray reaching task. The same analysis indicated a significant correlation (r = −0.71; p < 0.02) between dendritic length in the forelimb area and the number of contacts with the cylinder wall by ipsilateral paw and also a marginally significant correlation (r = −0.58; p < 0.06) between the same dendritic measure and the number of touches by contralateral paw in the cylinder task. Although, the correlation between dendritic length in the forelimb area and the number of strokes by ipsilateral paw in forepaw inhibition task was not significant (r = −0.24, p = 0.48), a significant correlation was found between dendritic length in the forelimb area and the number of strokes by contralateral paw (r = −0.66, p < 0.04). Finally, the correlation between the dendritic length in the forelimb area contralateral to the lesion side and consumption time in sunflower seed task was not significant (r = −0.45, p = 0.16) nor was the correlation between the dendritic length and number of shells in the task (r = 0.31, p = 0.34).
Although it is often believed that there is an inverse linear relationship between age-at-injury and functional recovery, the evidence from both rodent and carnivore studies does not support this idea [e.g., 11,32]. In rodents there appear to be clear windows of enhanced spontaneous recovery including the second week of life and late adolescence [e.g., 16,23]. Thus, for example, motor cortex injury on day 10 leads to better recovery than similar injury on days 1–5 or in adulthood [12]. Similarly, motor cortex injury on day 55 allows for better functional outcome than similar injury on day 35 or in adulthood [e.g., 23]. Curiously, the most extensive spontaneous recovery from motor cortex injury is after day 55 lesions as the day 10 lesions allow only partial recovery of skilled reaching. Similarly, animals with medial prefrontal lesions on day 10 also show chronic skilled reaching deficits although they show apparently normal performance on a variety of cognitive tasks [e.g., 11]. The current study confirms our previous findings that day 35 motor cortex lesions produce chronic deficits on a range of motor tasks and the performance levels in this study are virtually identical to those in our previous report [23]. The behavioral deficits are also very similar to those observed in animals with day 3 or adult motor cortex injuries [e.g., 12]. The reason for the age-dependent functional outcomes from similar motor cortex injuries at different times in development is not clear but presumably is related to the ongoing processes of brain development at the time of injury. It is likely that the mechanisms underlying the spontaneous recovery differ at different ages, however. During the second week of life there is extensive dendritic proliferation and the formation of astrocytes. These processes are largely complete well before age 55, a time that is likely characterized by dendritic pruning, although the data are pretty scarce. Lesions around days 7–12 produce enhanced gliogenesis and lead to increased dendritic arborization and/or spine density in pyramidal cells throughout the cerebral cortex [e.g., 14], and in some cases there is spontaneous neuronal proliferation as well [10,15]. Similarly, there is increased dendritic arborization and spine density after day 55 motor cortex lesions although no obvious evidence of enhanced neuronal or glial proliferation. The differences in neuronal reorganization after injury at different ages suggest that the brain may be most responsive to different types of rehabilitative treatments at different ages. This is because the treatments are working on a brain that is undergoing differing postinjury changes. This remains to be studied in detail, especially after injury in the juvenile or adolescent brain. Most studies designed to stimulate functional recovery after early injury has been on animals sustaining injuries prior to weaning. Finally, the enhanced dendritic arborization and spine density in the current study was regionally specific. Thus, there was an FGF-2 associated perilesional increase in dendritic length of the injured hemisphere, but not the intact one, as well as an increase in dendritic length in the intact forelimb region. The increase in the ipsilateral hemisphere is presumably related to the functional recovery but it is not so clear what role the contralateral dendritic changes might reflect. Eyre et al. [5] have shown that changes in
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the intact motor cortex of children with perinatal injuries may be related to the later development of cerebral palsy. A similar effect is unlikely here given that the animals showed such dramatic motor recovery. We have seen in parallel studies that increased dendritic arborization in the intact motor cortex is seen after neonatal hemidecortication but these animals still have severe motor deficits [13].
4.2. FGF-2 alters cerebral function and morphology after P35 motor cortex lesions We have previously shown that FGF-2 given either prenatally or postinjury stimulates functional recovery from medial prefrontal or posterior parietal injury on postnatal day 3 [e.g., 3]. Similarly, FGF-2 given postinjury stimulates functional recovery after day 10 motor cortex lesions. Administration of FGF-2 does not stimulate recovery from adult prefrontal or motor cortex lesions, but neutralizing antibodies to FGF-2 prevents spontaneous recovery from adult lesions [27]. The recovery seen in the current study differs from the benefits of FGF-2 after day 3 cortical injuries insofar as the level of recovery is more impressive in the current study – the treated animals performed all of the motor tasks as well as control animals in our previous studies. It is difficult to compare the extent of general recovery in our previous day 10 studies to the current one because the day 10 studies only measured skilled reaching. Nonetheless, in the skilled reaching task the effect of FGF-2 was equivalent after day 10 and day 35 lesions. One fundamental difference between the FGF-2 enhanced recovery after day 10, and 35 lesions is the effect of the treatment on cerebral morphology. FGF-2 stimulates extensive neuronal proliferation after day 10 injury, leading to a complete filling of the lesion region [20,21]. There was no obvious neuronal proliferation after FGF-2 administration on day 35 but there is enhanced dendritic arborization and spine density in the latter case. The mechanisms underlying the effect of FGF-2 administration are poorly understood but it is known that FGF-2 administration enhances both neuronal and glial proliferation [1,2,9,25]. FGF-2 is localized in certain neuron populations as well as astrocytes and FGF-2 production is upregulated after injury to the brain [e.g., 15], spinal cord [8], and peripheral nerves [4]. In addition, there is an increase in FGF-2 receptor expression following brain injury [26]. This latter effect is interesting because it may provide a route whereby the postinjury administration of FGF-2 can enhance reparative processes. A fundamental question, however, is just exactly what the changes in dendritic length and spine density actually mean. There are many studies showing that similar changes are correlated with motor learning so one possibility is that the changes reflect FGF2 enhanced motor learning, which in turn supports functional recovery. We do know that infusing antibodies to FGF-2 blocks spontaneous improvement in adults with similar motor cortex lesions and this is correlated with extensive dendritic stunting [27], which adds some support to the general idea. Furthermore, we did find correlations between several motor measures and dendritic length in the forelimb region. Just how the dendritic changes actually affect behavior are simply unknown at the present time, however, and will require detailed electrophysiological analysis to solve.
Acknowledgements This research was supported by an Alberta Heritage Foundation for Medical Research/Hotchkiss Brain Institute postdoctoral fellow-
ship to FN and a Canadian Institutes for Health Research grant to BK.
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Research report
FGF-2 induces behavioral recovery after early adolescent injury to the motor cortex of rats Farshad Nemati ∗ , Bryan Kolb Department of Neuroscience, University of Lethbridge, Lethbridge, AB, Canada, T1K 3M4
a r t i c l e
i n f o
Article history: Received 21 April 2011 Received in revised form 9 June 2011 Accepted 13 July 2011 Available online 23 July 2011 Keywords: Adolescence Motor cortex injury Basic fibroblast growth factor Dendritic reorganization Functional recovery
a b s t r a c t Motor cortex injuries in adulthood lead to poor performance in behavioral tasks sensitive to limb movements in the rat. We have shown previously that motor cortex injury on day 10 or day 55 allow significant spontaneous recovery but not injury in early adolescence (postnatal day 35 “P35”). Previous studies have indicated that injection of basic fibroblast growth factor (FGF-2) enhances behavioral recovery after neonatal cortical injury but such effect has not been studied following motor cortex lesions in early adolescence. The present study undertook to investigate the possibility of such behavioral recovery. Rats with unilateral motor cortex lesions were assigned to two groups in which they received FGF-2 or bovine serum albumin (BSA) and were tested in a number of behavioral tests (postural asymmetry, skilled reaching, sunflower seed manipulation, forepaw inhibition in swimming). Golgi–Cox analysis was used to examine the dendritic structure of pyramidal cells in the animals’ parietal (layer III) and forelimb (layer V) area of the cortex. The results indicated that rats injected with FGF-2 (but not BSA) showed significant behavioral recovery that was associated with increased dendritic length and spine density. The present study suggests a role for FGF-2 in the recovery of function following injury during early adolescence. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Basic fibroblast growth factor (FGF-2) plays a role in the regulation of cerebral development and in modulating recovery from cerebral injury [e.g., 3,6,18,20,21,27]. The effects of FGF-2 on functional recovery are especially large in animals with cortical injury in infancy, but the mechanisms appear to vary with age. Thus, animals with medial prefrontal lesions on postnatal day 3 show enhanced recovery that is associated with increased cortical thickness and dendritic arborization [e.g., 3]. In contrast, animals with motor cortex lesions on postnatal day 10 show enhanced motor recovery that is associated with the regeneration of the lost tissue [20–22]. This regeneration does not occur if the injuries are on day 3 or in adulthood or if the day 10 lesions are in more posterior cortex. The goal of the current study was to determine if FGF-2 would enhance recovery from motor cortex injuries in early adolescence either by stimulating the proliferation of new cells or the hypertrophy of cortical pyramidal cells. We have previously shown that rats given unilateral motor cortex lesions in early adolescence (postnatal day 35, P35) showed significant deficits on various motor tasks and no change in dendritic organization in perilesional or contralateral motor cortex
∗ Corresponding author. Tel.: +1 403 329 2044; fax: +1 403 329 2775. E-mail address: [email protected] (F. Nemati). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.07.023
pyramidal cells [23]. This result contrasted to the effects of late adolescent (P55) motor cortex lesions whereby animals showed nearly complete recovery associated with extensive dendritic hypertrophy. We therefore knew that adolescent rats were capable of significant functional recovery but that this did not happen if the injuries were in early adolescence. We reasoned that if early adolescent rats were capable of similar functional recovery, then it could be stimulated by postinjury application of FGF-2. The results confirmed our prediction. 2. Material and methods 2.1. Animals Female Long–Evans hooded rats were raised in the University of Lethbridge vivarium and group-housed in two or three individuals in different cages with bedding in the colony room. The temperature of the colony room maintained at 22 ◦ C on a 12 h light/dark cycle. The animals were assigned either to experimental or control groups in which they received basic fibroblast growth factor “FGF-2” (n = 5) or bovine serum albumin “BSA” (n = 6) respectively for seven consecutive days starting the day after placing motor cortex lesion in their left hemisphere. The University of Lethbridge Animal Care Committee approved experimental procedure in this study on behalf of the Canadian Council of Animal Care. We chose to use females in this study for two reasons. First, we used females in our previous study looking at P35 and P55 rats. Second, we elected to look at females because we had previously found that although FGF-2 did not affect most measures of recovery in a sexually-dimorphic manner, there was a differential effect on body weight [3]. Specifically, FGF-2 reduced body weight in both sham and lesion males but had no effect in females. Given that we were looking at motor behaviors and given that differences in body size can affect performance in motor tasks (and
F. Nemati, B. Kolb / Behavioural Brain Research 225 (2011) 184–191 especially the skilled reaching task because the bars are relatively further apart for smaller animals) we wanted to control for body weight differences. 2.2. Surgical procedures Unilateral motor cortex lesions were placed in the left hemisphere of the postnatal 35 (P35) day old rats. A standard procedure was followed to generate the lesions in which each animal was anesthetized in an induction chamber using 4% isoflurane first. Then rats were positioned in a stereotaxic device that was modified to fit the size of their body. During the surgery the animals were kept anesthetized with 2–3% isoflurane and the aspiration method was used to produce the lesions. In order to remove the motor cortex tissue an area of the skull corresponding to the anterior motor cortex was removed. The coordinates were from AP +2 to −2 and lateral from +1.5 to the temporal ridge. Following the removal of the dura, the tissue of motor neocortex was gently sucked out using a fine pipette. As soon as the lesion was completed and rats reached homeostasis the incision was sutured using the sterile 5-0 Vicryl suture. 2.3. FGF-2 injections FGF-2 diluted in BSA (0.1 M PO4 as a concentration of 1 mg/ml) at the concentration (1 g/ml) was injected s.c. at 0.1 ml/10 g of body weight. The concentration was demonstrated to be efficient in enhancing neurogenesis in P7–P28 in Wagner et al. [33] study, which was closest age to our sample (P35). Also, it has been shown that this concentration contributes to the recovery after motor cortex injury in developing rats [20].
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a corner of a Plexiglas box (45 cm × 14 cm × 35 cm) during each training or testing session. Animals were trained for four consecutive days and their behavior was video recorded on the last day. Two measures were used to quantify the behavior of animals: (1) time: the total amount of time spent on manipulating, opening the shell and consuming the seeds and (2) the number of pieces of shells left after animals retrieve and eat the seeds. If the pieces were too small to count, a maximum of 30 pieces of shells was assigned.
2.4.4. Forepaw inhibition swimming task Being semi-aquatic animals, rats only use their hind limbs for propulsion, while keeping their forelimbs immobile under their chin while swimming [19]. Motor cortex lesion is usually associated with the disinhibition of the forelimb (strokes) during the swimming in rats [17]. To evaluate the effect of FGF-2 treatment following motor cortex lesion on the swimming of the rats in this task the animals were trained for 4 days in a rectangular aquarium (120 cm × 43 cm × 50 cm). Rats had to swim from one end of the aquarium to a visible platform placed at the other end of the rectangular. Animals were released in the water (25 ◦ C) in one side of the rectangular to swim towards the platform on the other side on the training days. Each rat was given several consecutive trials (≈10 trials) to learn to swim strait without touching the wall of the pool. On the fourth day, animals’ behavior was video recorded while swimming to the platform. The dependent variable was the number of strokes during swimming to the platform without touching the wall.
2.5. Anatomical procedures 2.4. Behavioral procedures The behavioral tests (cylinder, tray reaching, sunflower seed, forepaw inhibition) began 1 month after surgery. In order to motivate the animals in the reaching and sunflower seed tests, rats were placed on a light food-restriction regime in which each rat obtained 20 g of food per day. Rats were fed 30 min after testing session every day. The body weight of the rats was monitored every day and maintained at ≈95–98% during the behavioral testing. 2.4.1. Cylinder test In order to measure the use of forelimb for weight support rats were tested in the cylinder test in which rats explore their environment vertically [28,29]. Each rat was placed in a transparent cylinder 20 cm in diameter and 30 cm high. The dependent variable was the number of contacts by each forepaw with the cylinder wall while simultaneous contact of limb with the wall of the cylinder was counted as a touch for each paw. Rats were placed gently in the cylinder to explore it for four consecutive days each day for 3 min. The animals’ explorative behavior inside the cylinder was videotaped on the last day. Using a mirror at an appropriate angle underneath the cylinder the experimenter was able to videotape the animals’ behavior from a ventral view. 2.4.2. Tray reaching task The forelimb movement skills were assessed in Tray reaching boxes that are designed to measure the natural behavior of food consumption in rats [19]. Each box was surrounded by clear Plexiglas on three sides and thin metal bars on the fourth side in the front. A food tray (4 cm wide and 0.5 cm deep) was positioned in front of the bars. In order to prevent the rats from dragging the chicken feed pellets into the box to consume them there was a small gap between the tray and testing box. The bottom of the test box was wired in a way that food pellet could fall through which and therefore rats could not pick up the food from the floor. In order to perform the task successfully rats had to reach and grasp the small pieces of food after passing the bars installed in front of the box, and skillfully retract their paws and consume the food. One month after surgery rats with motor cortex lesion in both BSA-injected and FGF2-injected group were trained in Tray reaching task for 10 consecutive days. The behavior of rats in the tray-reaching box was videotaped on the last day. The success percent was calculated according to the following formula:
success percent =
hit × 100 reach
Success percent was defined in terms of two variables: (1) hit defines a reaching movement in which a rat successfully inserts its paw through the bars, grasp the food pallet on the tray and eat the food in the box following retracting its paw; (2) reach includes both successful movements [hits] and unsuccessful movements [attempts]. 2.4.3. Sunflower seed task Rats use their limbs and digits during sunflower seed consumption [34]. In order to consume a seed rat first starts manipulating the seed and position it into an appropriate angle in relation to their mouth before shelling them. After rats chew a piece of shell away then they can split the seed into two halves longitudinally with a final bite [34]. In order to evaluate the animals’ ability to open and successfully consume seeds using their limbs and digits, five sunflower seeds were placed in
An overdose of sodium pentobarbital was given intracardially to each animal, which was perfused using 0.9% saline at the conclusion of behavioral tests. The brains of the animals were removed from the skull and immersed in 20 ml of Golgi–Cox solution. Following 14 days in Golgi–Cox solution the brains were transferred to 30% sucrose solution for 7 more days. The brains then were cut on a vibratome at 200 m [7], and the sections were mounted on 2% gelatin slides. The slides were stored in a humidity chamber until staining. Immediately after staining the brains were coverslipped using Permount.
2.5.1. Dendritic analysis The dendritic processes had to meet the following inclusive criteria to be selected as a sample for drawing procedure: (1) the dendritic processes should not be affected by stain precipitations, blood vessels, or heavy clusters of dendrites from other cells; (2) the apical and basilar dendrites must be visible on the section. The morphology of neurons was studied in three areas of neocortex: layer III of parietal area ipsilateral or contralateral to the lesion side and also layer V of forelimb area contralateral to the lesion side [35]. After drawing cells in the abovementioned area using the camera lucida the following measures were used in analyzing the structure of cells: total dendritic length, branch numbers, and spine density. In order to determine the branch numbers and total dendritic length, the neurons were drawn under camera lucida set for the magnification of 200×. Branches originating from the cell body were considered as basilar dendrites, and those originating from the main dendrite projected from the cell body as apical dendrites. The total length of dendrites was measured using Sholl’s [30] concentric ring procedure [31]. This method includes both the principal apical dendrite as well as all branches from it. The mean of total dendritic length (in m) was estimated through multiplying the mean total number of intersections by 20 m, which is the interval between concentric spheres from the center of the cell body. The numbers of spines on branch order of 2 or 3 of apical and that of 4 or 5 of basilar dendrites were counted using a camera lucida set for the magnification of 1000×. The density of spines is presented as the number of spines/10 m. We chose to make the spine measurements on these branch orders because we had seen effects of the injuries in these regions in our previous study.
2.5.2. Cortical thickness Golgi–Cox stained sections were projected on a Zeiss 2 POL projector set at a magnification of 13×. The thickness of motor neocortex was measured at medial, central and lateral points in the contralateral cortex at 5 Paxinos and Watson [24] planes: plane 10 (areas M1, S1J, AID); plane 18 (areas M1, S1BF, G1); plane 25 (areas M1, S1BF, G1); plane 37 (areas V2m, AuD, TeA); and, plane 43 (areas V2MM, V1B, TeA). Similar measurements were made at the central or lateral points on the lesion side. See Fig. 1 in Stewart and Kolb [31] for an illustration.
2.6. Statistics Repeated measure analysis of variance (ANOVA), and also unpaired t-test were used to analyze the data. Bonferroni t-test was used for post hoc analysis. Also, Pearson correlation was used to study the relationship between variables. Significant values have been illustrated by “*” to indicate p < 0.05 and by “**” to indicate p < 0.01.
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Fig. 1. Cylinder task. Rats in FGF-injected group made fewer numbers of contacts with the wall of the cylinder than rats in BSA-injected group. **p < 0.01.
3. Results 3.1. Behavioral analysis The behavior of rats assigned to FGF-2 or BSA groups was compared in four different motor behavior tasks (cylinder task, tray reaching test, sunflower seed task, forepaw inhibition swimming task) sensitive to limb and/or digit movements. In general, rats that received FGF-2 following unilateral motor cortex lesion in their left hemisphere at 35 days of age performed better in behavioral tasks as compared with those receiving BSA following the lesion. 3.1.1. Cylinder task While exploring the cylinder in a vertical position rats used their forelimb to support their weight against the cylinder wall. Rats in BSA group with motor cortex lesion generated at P35 touched the cylinder wall more frequently in comparison with rats in FGF-2 group with the same lesion. In general, the left forelimb (ipsilateral to the lesion side) was used more frequently to support animals’ weight against the wall than right forelimb (contralateral to the lesion side) regardless of animals’ group. Statistical analysis of the data confirms the observations. The following represents the formal analysis of data as illustrated in Fig. 1. The time spent on vertical exploration of the cylinder was not significantly different between groups (t[9] = 0.6, p = 0.55). Repeated measure ANOVA revealed a significant effect of group (F[1,9] = 11.51; p < 0.009) in the number of touches with the wall of the cylinder and a significant effect of limb use (F[1,9] = 6.08; p < 0.04) during the vertical exploration in which the limb ipsilateral to the lesion side was used more frequently. Also, there was no significant interaction of limb use by group (F[1,9] = 3.47; p = 0.09).
Fig. 2. Tray reaching task. Rats with motor cortex injuries that received FGF-2 were more successful in the reaching task than those in BSA-injected group. *p < 0.05.
injected group were more skillful than those in BSA-injected group and thus spent less time retrieving and consuming the seeds. Nevertheless, the number of pieces of the shells left after retrieval of the seeds was not significantly different between groups. Fig. 3 illustrates the performance of rats in the task. Unpaired t-test indicated a significant difference between groups (t[9] = 2.41, p < 0.04) in terms of time spent on retrieving and consuming seeds. The number of shells left after retrieval was not significantly different between groups (t[9] = 0.16, p = 0.87). 3.1.4. Forepaw inhibition swimming task Although one rat did not learn to swim towards the platform without touching the wall, others were matched in terms of the
3.1.2. Tray reaching test After 10 days of training in Tray reaching task, one rat in each group did not reach for food at all and was eliminated from the analysis. Two rats in the FGF-injected group reached for food with their ipsilateral paw while two other rats were ambidextrous. In BSA group one rat was ambidextrous and other rats used their ipsilateral paw when reaching. Rats with unilateral motor cortex lesion at P35 demonstrated more successful reaching behavior following FGF-2 injections in comparison with rats that received their lesion at P35 but were injected BSA as shown in Fig. 2. Unpaired t-test indicated a significant difference between groups (t[7] = 2.38, p < 0.05). 3.1.3. Sunflower seed task Following 3 days of training in sunflower seed task, the behavior of rats were video recorded on the following day. Rats in FGF2-
Fig. 3. Sunflower seed task. (A) Rats in both FGF-2 and BSA-injected groups demonstrated almost the same level of skill in shelling the seeds measured in terms of the pieces of shells left. (B) Rats in FGF-2 group spent shorter period of time shelling and consuming the seeds than those in the BSA-injected group. *p < 0.05.
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no significant effect of forelimb use in terms of the number of strokes (F[1,8] = 1.86; p = 0.2), and no significant interaction of forelimb use by group (F[1,8] = 2.99; p = 0.12).
3.2. Histological results The lesions included the entire forelimb region of the left cerebral cortex as well as much of the body and some of the hindlimb region as illustrated in Fig. 5. No direct injury to the underlying striatum was observed.
3.2.1. Brain weight Unpaired t-test indicated no significant difference (t[9] = 1.93, p = 0.08) between groups in terms of the brain weight. Fig. 4. Forepaw inhibition task. Rats in FGF-2 group made fewer strokes during the swimming than those in BSA group. **p < 0.01.
number of trials that they were able to swim towards the platform without touching the wall of the aquarium during the videotaping. Rats in FGF2-injected group showed dramatic improvement in their swimming towards the platform as compared with the rats in BSA-injected group as shown in Fig. 4. Repeated measure ANOVA indicated a significant effect of group (F[1,8] = 11.47; p < 0.01), but
3.2.2. Cortical thickness The thickness of cortex was measured in the medial, central, and lateral points in the hemisphere contralateral to the lesion. Because the lesion had expanded towards the midline suture in the lesion side the thickness of the cortex was measured only at the central and lateral points in the lesion hemisphere. There was no group effect of cortical thickness in either hemisphere.
Fig. 5. Serial drawings of coronal sections of Golgi–Cox-stained tissues of the brains with motor cortex lesions. Brains from each group were ranked by lesion size and the median lesions were chosen as representative examples.
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Fig. 6. Cortical thickness at the medial point contralateral to the lesion hemisphere. The histobars from right to left in each group (BSA or FGF) corresponds to the plane 10 (areas M1, S1J, AID); plane 18 (areas M1, S1BF, G1); plane 25 (areas M1, S1BF, G1); plane 37 (areas V2m, AuD, TeA); and, plane 43 (areas V2MM, V1B, TeA). A dramatic increase in the thickness of the cortex from plane 18 (areas M1, S1BF, G1) to plane 25 (areas M1, S1BF, G1) was observed in FGF-2 injected rats compared to those in BSA-injected group. *p < 0.05.
3.2.3. Ipsilateral cortical thickness ANOVA indicated that the measures of cortical thickness in the central sections ipsilateral to the lesion are not significantly different (F[1,9] = 0.11; p = 0.74) between groups. There was a significant effect of planes (F[4,36] = 10.12; p < 0.0002), but no significant interaction of group by planes (F[4,36] = 0.49; p = 0.73). The plane effect reflects the normal thinning of the cortex in a rostral–caudal direction. The group effect was not significant (F[1,9] = 0.13; p = 0.72) in the lateral points, but a significant effect of thickness (F[1,9] = 18.60; p < 0.0002) was found in planes of the central points representing the normal thinning going from anterior to posterior cortex. There was no interaction of plane by group (F[4,36] = 2.43; p = 0.06). 3.2.4. Contralateral cortical thickness ANOVA indicated no significant difference between groups in cortical thickness of medial section contralateral to the lesion side (F[1,9] = 0.002; p = 0.96). There was a significant difference between the cortical thickness in different planes (F[4,36] = 39.10; p < 0.0002) that were in interactions with the group (F[4,36] = 2.75; p < 0.05). Post hoc analysis indicated a significant increase in the thickness of the cortex from plane 18 (areas M1, S1BF, G1) to plane 25 (areas M1, S1BF, G1) in FGF-2 injected rats compared to those in BSA-injected group (Fig. 6). ANOVA also indicated no significant effect of group in the measures of cortical thickness in the central section of contralateral hemisphere (F[1,9] = 1.50; p = 0.25). There was a significant effect of planes (F[4,36] = 21.83; p < 0.0002) that were in no interaction with the group (F[4,36] = 1.07; p = 0.38). No significant difference was found between groups in cortical thickness of the lateral section (F[1,9] = 0.02; p = 0.89). There was a significant difference between the cortical thickness of different planes (F[4,36] = 27.88; p < 0.0002), with no significant interaction by group (F[4,36] = 2.16, p = 0.09). 3.2.5. Morphological analysis of neurons in parietal cortex (layer III) 3.2.5.1. Dendritic length (Sholl analysis). The Golgi analysis demonstrated that the total length of basilar dendrites in FGF-2 group was longer than those that received BSA injection after receiving the same lesion at the same age (P35). The total length of apical dendrite of neurons was not significantly different between groups.
Fig. 7. Dendritic length of neurons (in m) in parietal cortex (III). The total length of dendrites in apical field (A) was virtually the same in both FGF-2 and BSA-injected groups, but in the basilar field (B) it was greater in rats that received FGF-2 injections than those in BSA-injected group. *p < 0.05.
The following section represents the formal analysis of the results illustrated in Fig. 7. A two way ANOVA revealed a significant effect of group (F[1,9] = 6.24; p < 0.04) in dendritic length of basilar dendrites of pyramidal neurons in layer III of parietal cortex. There was no significant effect of hemispheres (F[1,9] = 1.01; p = 0.34) nor an interaction of hemispheres by group (F[1,9] = 0.43; p = 0.52). In contrast, ANOVA on apical dendrite found no significant effect of group (F[1,9] = 1.12; p = 0.31) hemisphere (F[1,9] = 0.10; p = 0.75), or the interaction (F[1,9] = 0.04; p < 0.84) in the length of apical dendrites.
3.2.5.2. Branch order. There were no differences in the number of basilar or apical dendritic branches in parietal cortex (layer III). A two way ANOVA (treatment × hemisphere) indicated no significant effect of either treatment in branch number of basilar dendrites (F[1,9] = 2.84; p = 0.12) hemisphere (F[1,9] = 0.46; p = 0.51) or the interaction of hemisphere by group (F[1,9] = 0.79; p = 0.39). Similarly, ANOVA found no significant effect of treatment, hemisphere or the interaction for the apical dendrites (F[1,9] = 0.42; p = 0.53, F[1,9] = 0.26; p = 0.61; F[1,9] = 0.09; p = 0.76).
3.2.5.3. Spine density. There was an increase in spine density of the basilar, but not apical, dendrites in the ipsilateral, but not in the contralateral hemisphere, in the FGF-2 group relative to the BSA group (Fig. 8). A two-way ANOVA revealed no group effect in spine density in the basilar dendrite (F[1,9] = 4.21; p = 0.07) but there was a significant effect of hemisphere (F[1,9] = 10.26; p < 0.02) and the group × hemisphere interaction (F[1,9] = 9.41; p < 0.02). In contrast, there were no significant effects of group, hemisphere, or the interaction for the apical dendrites: (F[1,9] = 0.97; p = 0.35, F[1,9] = 0.24; p = 0.63, F[1,9] = 2.79; p = 0.12).
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4. Discussion The two primary findings of the current experiment were: (1) there is continuing evidence for age-dependent patterns of functional recovery after cortical injury during development; and (2) the administration of FGF-2 alters cerebral function and morphology after P35 motor cortex lesions. We consider each finding separately. 4.1. There are age-dependent effects of cortical injury during development
Fig. 8. Spine density (per 10 m) in parietal cortex (III). The density of spines in basilar field of parietal cortex ipsilateral (I) but not contralateral (C) to the lesion side was greater in FGF-2 group than those in BSA-injected group. *p < 0.05.
3.2.6. Morphological analysis of basilar fields of neurons in contralateral forelimb area of cortex (layer V) 3.2.6.1. Dendritic length. The total length of dendrites was longer in rats with motor cortex lesion treated with FGF-2 than those in BSAinjected group (M’s = 1709.6 ± 210.33; M’s = 1214.66 ± 110.55). Statistical analysis of data using unpaired t-test indicated a marginally significant difference between groups (t[9] = 2.19; p < 0.06) in dendritic length of basilar field in layer V neurons of forelimb area of the cortex. 3.2.6.2. Branch order. The number of branches in contralateral hemisphere of the brain was almost the same in both groups (M’s = 28.96 ± 3.63; M’s = 23.03 ± 1.93). Unpaired t-test found no significant effect of group in branch order (t[9] = 1.51; p = 0.16). 3.2.6.3. Spine density. Although there was a trend for an increase in spine density in the FGF-2 compared to BSA group (M’s = 9.54 ± 0.64; M’s = 8.15 ± 0.35), the effect was not significant (t[9] = 1.97; p = 0.08). 3.3. Correlations Pearson correlation was used to study the relationship between the behavioral measures and reorganized dendritic measure (dendritic length) of forelimb cortex (V) as an area responsible for direct control of forepaw movements. The analyses indicated a meaningful relationship between dendritic length in the forelimb area contralateral to the lesion side and motor behaviors. The analysis revealed a significant correlation (r = 0.67; p < 0.05) between dendritic length in the forelimb area contralateral to the lesion side and success in tray reaching task. The same analysis indicated a significant correlation (r = −0.71; p < 0.02) between dendritic length in the forelimb area and the number of contacts with the cylinder wall by ipsilateral paw and also a marginally significant correlation (r = −0.58; p < 0.06) between the same dendritic measure and the number of touches by contralateral paw in the cylinder task. Although, the correlation between dendritic length in the forelimb area and the number of strokes by ipsilateral paw in forepaw inhibition task was not significant (r = −0.24, p = 0.48), a significant correlation was found between dendritic length in the forelimb area and the number of strokes by contralateral paw (r = −0.66, p < 0.04). Finally, the correlation between the dendritic length in the forelimb area contralateral to the lesion side and consumption time in sunflower seed task was not significant (r = −0.45, p = 0.16) nor was the correlation between the dendritic length and number of shells in the task (r = 0.31, p = 0.34).
Although it is often believed that there is an inverse linear relationship between age-at-injury and functional recovery, the evidence from both rodent and carnivore studies does not support this idea [e.g., 11,32]. In rodents there appear to be clear windows of enhanced spontaneous recovery including the second week of life and late adolescence [e.g., 16,23]. Thus, for example, motor cortex injury on day 10 leads to better recovery than similar injury on days 1–5 or in adulthood [12]. Similarly, motor cortex injury on day 55 allows for better functional outcome than similar injury on day 35 or in adulthood [e.g., 23]. Curiously, the most extensive spontaneous recovery from motor cortex injury is after day 55 lesions as the day 10 lesions allow only partial recovery of skilled reaching. Similarly, animals with medial prefrontal lesions on day 10 also show chronic skilled reaching deficits although they show apparently normal performance on a variety of cognitive tasks [e.g., 11]. The current study confirms our previous findings that day 35 motor cortex lesions produce chronic deficits on a range of motor tasks and the performance levels in this study are virtually identical to those in our previous report [23]. The behavioral deficits are also very similar to those observed in animals with day 3 or adult motor cortex injuries [e.g., 12]. The reason for the age-dependent functional outcomes from similar motor cortex injuries at different times in development is not clear but presumably is related to the ongoing processes of brain development at the time of injury. It is likely that the mechanisms underlying the spontaneous recovery differ at different ages, however. During the second week of life there is extensive dendritic proliferation and the formation of astrocytes. These processes are largely complete well before age 55, a time that is likely characterized by dendritic pruning, although the data are pretty scarce. Lesions around days 7–12 produce enhanced gliogenesis and lead to increased dendritic arborization and/or spine density in pyramidal cells throughout the cerebral cortex [e.g., 14], and in some cases there is spontaneous neuronal proliferation as well [10,15]. Similarly, there is increased dendritic arborization and spine density after day 55 motor cortex lesions although no obvious evidence of enhanced neuronal or glial proliferation. The differences in neuronal reorganization after injury at different ages suggest that the brain may be most responsive to different types of rehabilitative treatments at different ages. This is because the treatments are working on a brain that is undergoing differing postinjury changes. This remains to be studied in detail, especially after injury in the juvenile or adolescent brain. Most studies designed to stimulate functional recovery after early injury has been on animals sustaining injuries prior to weaning. Finally, the enhanced dendritic arborization and spine density in the current study was regionally specific. Thus, there was an FGF-2 associated perilesional increase in dendritic length of the injured hemisphere, but not the intact one, as well as an increase in dendritic length in the intact forelimb region. The increase in the ipsilateral hemisphere is presumably related to the functional recovery but it is not so clear what role the contralateral dendritic changes might reflect. Eyre et al. [5] have shown that changes in
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the intact motor cortex of children with perinatal injuries may be related to the later development of cerebral palsy. A similar effect is unlikely here given that the animals showed such dramatic motor recovery. We have seen in parallel studies that increased dendritic arborization in the intact motor cortex is seen after neonatal hemidecortication but these animals still have severe motor deficits [13].
4.2. FGF-2 alters cerebral function and morphology after P35 motor cortex lesions We have previously shown that FGF-2 given either prenatally or postinjury stimulates functional recovery from medial prefrontal or posterior parietal injury on postnatal day 3 [e.g., 3]. Similarly, FGF-2 given postinjury stimulates functional recovery after day 10 motor cortex lesions. Administration of FGF-2 does not stimulate recovery from adult prefrontal or motor cortex lesions, but neutralizing antibodies to FGF-2 prevents spontaneous recovery from adult lesions [27]. The recovery seen in the current study differs from the benefits of FGF-2 after day 3 cortical injuries insofar as the level of recovery is more impressive in the current study – the treated animals performed all of the motor tasks as well as control animals in our previous studies. It is difficult to compare the extent of general recovery in our previous day 10 studies to the current one because the day 10 studies only measured skilled reaching. Nonetheless, in the skilled reaching task the effect of FGF-2 was equivalent after day 10 and day 35 lesions. One fundamental difference between the FGF-2 enhanced recovery after day 10, and 35 lesions is the effect of the treatment on cerebral morphology. FGF-2 stimulates extensive neuronal proliferation after day 10 injury, leading to a complete filling of the lesion region [20,21]. There was no obvious neuronal proliferation after FGF-2 administration on day 35 but there is enhanced dendritic arborization and spine density in the latter case. The mechanisms underlying the effect of FGF-2 administration are poorly understood but it is known that FGF-2 administration enhances both neuronal and glial proliferation [1,2,9,25]. FGF-2 is localized in certain neuron populations as well as astrocytes and FGF-2 production is upregulated after injury to the brain [e.g., 15], spinal cord [8], and peripheral nerves [4]. In addition, there is an increase in FGF-2 receptor expression following brain injury [26]. This latter effect is interesting because it may provide a route whereby the postinjury administration of FGF-2 can enhance reparative processes. A fundamental question, however, is just exactly what the changes in dendritic length and spine density actually mean. There are many studies showing that similar changes are correlated with motor learning so one possibility is that the changes reflect FGF2 enhanced motor learning, which in turn supports functional recovery. We do know that infusing antibodies to FGF-2 blocks spontaneous improvement in adults with similar motor cortex lesions and this is correlated with extensive dendritic stunting [27], which adds some support to the general idea. Furthermore, we did find correlations between several motor measures and dendritic length in the forelimb region. Just how the dendritic changes actually affect behavior are simply unknown at the present time, however, and will require detailed electrophysiological analysis to solve.
Acknowledgements This research was supported by an Alberta Heritage Foundation for Medical Research/Hotchkiss Brain Institute postdoctoral fellow-
ship to FN and a Canadian Institutes for Health Research grant to BK.
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