Prolonged exercise induces angiogenesis and increases cerebral blood volume in primary motor cortex of the rat

Neuroscience 117 (2003) 1037–1046

PROLONGED EXERCISE INDUCES ANGIOGENESIS AND INCREASES CEREBRAL BLOOD VOLUME IN PRIMARY MOTOR CORTEX OF THE RAT R. A. SWAIN,a,h* A. B. HARRIS,g,i E. C. WIENER,b,h,i M. V. DUTKA,h H. D. MORRIS,i B. E. THEIEN,h S. KONDA,c,h,i K. ENGBERG,h P. C. LAUTERBURf,g,h,i AND W. T. GREENOUGHa,d,e,f,h

forelimb regions of motor cortex. FAIR images acquired during experiment 2 under normocapnic and hypercapnic conditions indicated that resting cerebral blood flow was not altered under normal conditions but was elevated in response to high levels of CO2, suggesting that prolonged exercise increases the size of a capillary reserve. Finally, the immunohistological data indicated that exercise induces robust growth of capillaries (angiogenesis) within 30 days from the onset of the exercise regimen. Analysis of other regions failed to find any changes in perfusion or capillary structure suggesting that this motor activity-induced plasticity may be specific to motor cortex. These data indicate that capillary growth occurs in motor areas of the cerebral cortex as a robust adaptation to prolonged motor activity. In addition to capillary growth, the vascular system also experiences heightened flow under conditions of activation. These changes are chronic and observable even in the anesthetized animal and are measurable using noninvasive techniques. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.

a Department of Psychology, University of Illinois, Urbana, IL 61801, USA b Department of Nuclear Engineering, University of Illinois, Urbana, IL 61801, USA c

Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL 61801, USA

d Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA e Department of Psychiatry, University of Illinois, Urbana, IL 61801, USA f

Neuroscience Program, University of Illinois, Urbana, IL 61801, USA

g

Program in Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA h

Beckman Institute, University of Illinois, Urbana, IL 61801, USA

i

Biomedical Magnetic Resonance Laboratory (Department of Medical Information Science, College of Medicine), University of Illinois, Urbana, IL 61801, USA

Key words: capillary, fMRI, vascular.

CNS plasticity is not restricted to modifications of neuronal connectivity and synaptic morphology. While studies of experience-dependent changes in synapse number and morphology have constituted the bulk of work in the field (e.g. Greenough et al., 1994), it has recently become clear that nonneuronal elements (e.g. glia and capillaries) can exhibit a similar capacity for plastic change. For example, transient changes in brain blood flow and volume have been associated with sensory, motor, and cognitive task performance (Ogawa et al., 1990, 1992, 1993; Kwong et al., 1992; Kim et al., 1993; Bandettini et al., 1994; Binder et al., 1994; Cao et al., 1994; Burdett et al., 1995). These responses are sustained for only a few seconds following termination of the task. In contrast, long-term increases in capillary number have been detected in the rat visual cortex following rearing in a complex environment (EC), relative to counterparts housed individually in cages (inactive control [IC]) (Black et al., 1987). The EC environment promotes growth in overall cortical weight and thickness (Diamond et al., 1964). This growth involves increases in the number of dendritic branches and synapses per neuron in EC rats relative to their IC littermates (Greenough and Volkmar, 1973; Sirevaag and Greenough, 1985; Turner and Greenough, 1985). These neuronal changes are accompanied by substantial increases in glia and capillary volume. The capillary change is especially robust. Weanling rats reared in EC exhibit a near doubling of

Abstract—Plastic changes in motor cortex capillary structure and function were examined in three separate experiments in adult rats following prolonged exercise. The first two experiments employed T-two-star (T2*)-weighted and flow-alternating inversion recovery (FAIR) functional magnetic resonance imaging to assess chronic changes in blood volume and flow as a result of exercise. The third experiment used an antibody against the CD61 integrin expressed on developing capillaries to determine if motor cortex capillaries undergo structural modifications. In experiment 1, T2*-weighted images of forelimb regions of motor cortex were obtained following 30 days of either repetitive activity on a running wheel or relative inactivity. The proton signal intensity was markedly reduced in the motor cortex of exercised animals compared with that of controls. This reduction was not attributable to alterations of vascular iron levels. These results are therefore most consistent with increased capillary perfusion or blood volume of *Correspondence to: R. A. Swain, 208 Garland Hall, Department of Psychology, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA. Tel: ⫹1-414-229-5329; fax: ⫹1-414-229-5321. E-mail address: [email protected] (R. A. Swain). Abbreviations: ABC, avidin-biotin complex; ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; BG, background; CPV, converted pixel value; DAB, 3,3'-diaminobenzidine; EC, complex environment; FAIR, flow alternating inversion recovery; FMRI, functional magnetic resonance imaging; HS, horse serum; IC, inactive control; IgG, immunoglobulin; MC, motor cortex; MR, magnetic resonance; N, noise; OC2MM, occipital cortex; area 2, mediomedial; OD, optical density; PB, phosphate buffer; PBS, phosphate buffered saline; PV, pixel value; ROI, region of interest; SNR, signal to noise ratio; T2*, T-two-star; TE, echo time; TI, time period following the preparatory inversion; TR, repetition time; VX, voluntary exercise.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00664-4

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capillary volume per neuron in visual cortex relative to ICs (Black et al., 1987). The relative contributions of motor-skill learning and motor activity to brain plasticity cannot be distinguished in the EC paradigm. To assess those contributions, Black et al. (1990) developed a paradigm in which the morphological sequelae of motor activity and motor-skill learning could be dissociated. In their paradigm, rats were trained to traverse a series of obstacles that required a substantial degree of motor learning to master but which necessitated only minimal motor activity relative to animals that were housed with ad libitum access to running wheels. Examination of the cerebellar paramedian lobule revealed a 25% increase in the number of synapses per Purkinje cell in motor-learning animals but no changes in capillary density. By contrast, running-wheel animals exhibited angiogenesis but not synaptogenesis. These results suggest two very different patterns of brain adaptation to environmental demands: acquisition of new skills involves the formation of new circuitry while extensive activation of existing circuits requires the addition of new vasculature. In this report, we describe three experiments in which we explored cerebral blood flow and angiogenesis in motor cerebral cortex as well as non-motor sites of the rat brain. Our primary goal was to ascertain whether primary motor cortex (MC) displays capillary changes similar to those reported for cerebellum following prolonged motor activity (Black et al., 1990) and to establish if the elevated levels of neural activation associated with exercise produce chronic changes in localized measures of cerebral blood flow. To determine whether resting cerebral blood volume is affected by activity, in experiment 1 we examined the MC of rats undergoing prolonged physical exercise and associated neural activation using noninvasive functional magnetic resonance imaging (fMRI). The validity of these methods relies on the demonstration that there exists a coupling between vascular perfusion and neuronal activation (Sokoloff, 1977; Sokoloff et al., 1977; Fox and Raichle, 1986; Grinvald et al., 1986). T2*-weighted imaging is sensitive to physiological processes that distort the magnetic field. These processes include changes in ferritin concentration or blood oxygenation. T2*-weighted imaging uses deoxyhemoglobin as an endogenous contrast agent (Ogawa et al., 1990, 1992, 1993; Kwong et al., 1992; Kim et al., 1993; Bandettini et al., 1994; Binder et al., 1994; Cao et al., 1994; Burdett et al., 1995). Because deoxyhemoglobin is paramagnetic, microscopic distortions in the magnetic field will occur in the vicinity of blood cells and vessels containing deoxyhemoglobin, leading to a local signal deficit. Any factor that modulates the deoxyhemoglobin concentration within a region of the brain, i.e. blood flow change, blood volume change, or change in oxygen extraction, will in turn cause a modulation of the local signal. T2*-weighted imaging has been successfully used in animals to detect transient hypercapnia effects on perfusion and in humans to detect visual and auditory cortical responses to sensory stimulation, brain activation by other modalities and long-term effects of brain injury on transient MC activation patterns (Ogawa et al., 1990, 1992, 1993;

Kwong et al., 1992; Kim et al., 1993; Bandettini et al., 1994; Binder et al., 1994; Cao et al., 1994; Burdett et al., 1995). To determine whether any effect of activity was specific to motor regions of the cerebral cortex, we also examined non-motor regions of frontoparietal cortex as well as motor and non-motor subcortical structures (striatum and medial septal nucleus respectively). Given that a number of hemodynamic factors could contribute to any difference in signal intensity observed in the T2* experiment presented above, we decided in experiment 2 to utilize a second fMRI technique, flow alternating inversion recovery (FAIR), which is sensitive to blood flow, to separate these variables. FAIR is a continuous-pulse technique in which two inversion recovery images are acquired, one with a non-selective preparatory inversion pulse and one with a selective preparatory inversion pulse inverting only those spins within the imaged slice. During the time period following the preparatory inversion (TI), fully relaxed spins will flow into the slice and exchange with inverted spins in the case of a selective inversion and in the case of a non-selective inversion, in-flowing inverted spins will exchange with already inverted spins within the slice with no net effect (Kim, 1995). The former case is sensitive to flow while the latter is insensitive. Subtraction of the images provides a measure of microvascular blood flow (Kwong et al., 1995). In the present experiment, images were collected under both basal and hypercapnic conditions in both voluntary exercise (VX) and IC rats. Hypercapnia has previously been demonstrated to significantly increase cerebral blood flow (Phillis, 1993). Experiment 3 was conducted in order to determine if any chronic changes in capillary function observed in the earlier experiments were also accompanied by changes in the structure of this system. In this experiment, we used an antibody, F11, which recognizes rat CD61, the ␤3 subunit of the ␣v␤3 integrin. Integrin ␣v␤3 promotes endothelial-cell interactions with extracellular matrix components (Cheresh, 1987, 1991; Hynes, 1992). In addition to its adhesive roles, ␣v␤3 also initiates a calcium-dependent signal which may be important for endothelial-cell motility (Leavesley et al., 1993). It is expressed at high levels on blood vessels during development or in adults during wound repair (Brooks et al., 1994a). Application of an ␣v␤3 antagonist during active blood vessel growth in living tissue produces apoptosis of the growing vessel while sparing mature, non-budding capillaries (Brooks et al., 1994b). In the non-damaged, mature vessel, the ␤3 subunit has bound to fibronectin and is thus unavailable for labeling. Therefore, any labeling observed in experiment 3 can be presumed to be on new, developing capillaries.

EXPERIMENTAL PROCEDURES Experiment 1 Subjects. All animals in the following experiments were maintained, managed, and cared for according to the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication number 80-23). The number of rats used in each experiment reflects the minimum number of animals that were necessary to achieve reliable statistical accuracy. No significant discomfort or

R. A. Swain et al. / Neuroscience 117 (2003) 1037–1046 pain was experienced by any animal during the course of these experiments. Nine litter-matched same-sex pairs of male and female LongEvans hooded rats (Rattus norvegicus) from the Beckman Institute breeding colony participated in the first part of this experiment. The animals’ ages ranged from 6 months to 1 year of age. Prior to the onset of the experiment, each littermate pair was housed together in a standard laboratory cage and maintained under a 12-h light/dark schedule. All rats were allowed ad libitum access to food and water. Fourteen additional female animals were used for the second part of this experiment. All animals were of comparable age and experienced similar training to those reported above. Training and imaging parameters. Each rat was assigned to either a VX (running wheel; circumference⫽111.8 cm) or IC (standard laboratory cage) group. The rats were allowed at least 30 days of this differential experience before magnetic resonance (MR) imaging. For the first portion of this experiment, we determined whether prolonged exercise alters blood perfusion of MC. One littermate pair was imaged per night. The order of imaging was counterbalanced across sessions. All sessions began 3 h prior to the commencement of the animals’ dark cycle. Under anesthesia (ketamine [65 mg/kg]⫹xylazine [8 mg/kg]⫹acepromazine [5 mg/kg]), each rat was inserted into a 4.7T/33-cm-bore magnet of an MR imaging system (SISCO, Palo Alto, CA, USA). High-resolution anatomical images (256⫻128 spin-echo image; repetition time [TR]⫽3.6 s; echo time [TE]⫽45 ms; slice thickness⫽0.156 mm⫻0.312 mm in plane resolution) were obtained in the coronal plane. From these images, the coordinates of the slice with the largest forelimb representation (based on local cortical and subcortical landmarks) was determined for each rat and it was at these coordinates that all subsequent images were obtained (approximately 0.20 mm anterior to bregma). To minimize the possibility that differing depths of anesthesia might contribute to any differences in observed signal intensity, precisely 1 h after the initial anesthetization, 128⫻128 2D gradient-echo images (1.6-mm slice thickness; TR⫽.1 s; TE⫽10 ms; flip angle⫽15°; four transients; 0.312 mm⫻0.312 mm in plane resolution) were collected. Ferritin and iron labeling. For the second part of this experiment, we determined whether basal capillary levels of iron and ferritin were modulated by behavioral activity. Ferritin is heterogeneously distributed in the brain and can be localized to perivascular cells (Connor and Benkovic, 1992; Connor et al., 1994; Connor and Menzies, 1995). Increased incorporation of iron or ferritin in the cerebrovascular system as a result of repetitive behavioral activity would seriously compromise our interpretation of any T2* effect since these metals would depress proton signal intensity. Therefore, seven VX and seven IC rats, trained identically to those above, were killed and perfused with 0.1-M phosphate buffer (PB; 7.3–7.4 pH) followed by fixation with 4% paraformaldehyde in PB. The brains were removed, cryoprotected in 30% sucrose in PB, and then sectioned into 52-␮m coronal sections. Sections were randomly chosen from motor and visual cortices for subsequent staining. Eighteen sections containing the MC and 18 sections containing the visual cortex and hippocampus were selected from each animal. The tissue sections were then washed twice in phosphate buffered saline (PBS) and placed in a 0.3% hydrogen peroxide solution for 60 min. Next, the sections were washed twice in PBS and twice in a 2% solution of goat serum in PBS and blocked overnight at 4 °C in a solution of 10% serum and 0.5% Triton-X100 in PBS. Following two PBS washes and two serum washes the next day, the tissue was incubated overnight at 4 °C in a primary antibody solution (1% serum, 0.5% Triton-X, and a 1:2000

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dilution of anti-horse spleen ferritin raised in rabbit [Sigma] in PBS). On day 3, the tissue was washed as before and then incubated for 90 min at room temperature in the secondary antibody solution (10% goat serum and a 1:500 dilution of biotinylated anti-rabbit immunoglobulin G [IgG] developed in goat [Sigma] in PBS). Next, the slices were washed twice in PBS before being transferred to an avidin– biotin complex (ABC) solution for 60 min at room temperature. After this incubation, the sections were washed as before in PBS and then in Tris–HCl buffer (twice in each). The tissue was then placed into a 3,3'-diaminobenzidine (DAB) solution (100 mg DAB, 200 ml Tris–HCl buffer, 1.39 g nickel ammonium sulfate, and 20 ␮l of 30% hydrogen peroxide) until staining occurred (approximately 30 s to 1 min). The tissue was then immediately washed five times in PBS. Finally, the sections were mounted on gelled slides, dried overnight, dehydrated, cleared, and coverslipped with Permount. For each animal, one section was used as an immunohistochemical control, which included the following: 1) incubation with buffer excluding the primary antibody, 2) incubation in the ABC and, 3) incubation in DAB. The results of these negative controls were consistently negative. In addition, the presence of iron was detected using Perl’s DAB staining. Sixteen sections containing the MC and 16 sections containing the visual cortex and hippocampus were selected from five VX and six IC animals (three animals were eliminated due to poor-quality staining). The sections were mounted on slides and stained for 30 min with Prussian Blue (1% potassium ferrocyanide and 7.4% HCl). The slides were then rinsed in deionized water for 10 min. For staining intensification, the slides were placed into a DAB solution (1.5 g DAB in 300 ml PB) for 15 min. The slides were removed and then placed back into the DAB solution for 90 min after addition of 50 ␮l of 30% hydrogen peroxide. Finally, the slides were rinsed in deionized water for 10 min, allowed to dry, dehydrated, cleared, and coverslipped with Permount. Staining intensity of both the ferritin- and iron-labeled sections was determined in a double-blind fashion. Digital images of the slices were analyzed for pixel values using NIH Image. Five samples per region of interest were obtained in each slice. The data for each animal were averaged to obtain one mean pixel value (PV) per animal for each staining procedure. PV data were converted to optical density (OD) using OD⫽1.0009*CPV [converted PV]⫹0.00002 where CPV⫽0.7394 log10 (60,000/PV⫺275) (Green, 1998). The forelimb MC OD was normalized to the visual cortex OD in both the VX and IC groups. A two-way analysis of variance (ANOVA) was performed on each set of data using group (VX and IC) and structure (dentate gyrus and MC) as the independent variables and normalized OD as the dependent measure.

Experiment 2 Subjects. Five litter-matched pairs of Long-Evans hooded rats (Rattus norvegicus) participated in this experiment. All animals were females and approximately 6 months old. Prior to the onset of the experiment, each littermate pair was housed together in a standard laboratory cage and maintained under a 12-h light/ dark schedule. All rats were allowed ad libitum access to food and water during the course of behavioral training. Twelve hours prior to imaging, food intake was terminated to lessen the severity of nausea associated with hypercapnia. Training and imaging parameters. Each rat was assigned to either a VX (running wheel; circumference⫽111.8 cm) or IC (standard laboratory cage) group. The rats were allowed at least 30 days of this differential experience before MR imaging. One VX/IC pair was imaged per day. Immediately prior to imaging, each rat was administered an i.m. cocktail of ketamine (65 mg/kg), xylazine (8 mg/kg), and acepromazine (5 mg/kg). Body temperature was maintained by placing the animal on an isothermal pad. Each animal was then inserted into a 4T SMIS/ Magnex animal imaging system. Coronal sections of primary MC

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were acquired using an inversion recovery spin echo sequence (TR⫽2.8s, TI⫽1.4s, TE⫽18ms). The inversion pulse was an 8-ms hyperbolic secant pulse applied with and without a slice-select gradient, this gradient conferring the flow sensitivity, for alternating images. The minimum inversion thickness required to fully invert the imaging slice during the slice-selective inversion was experimentally determined to be three times the imaging-slice thickness. The images were collected at both basal and hypercapnic conditions. During hypercapnia, rats breathed a 10% CO2/90% air mixture. Two reference images (128⫻128), one flow sensitive under hypercapnic conditions, and one flow insensitive, were acquired. Sixteen low-resolution (32⫻128) images with alternating flow sensitivity were then collected. Four averages were acquired for each image. Hypercapnic and normocapnic conditions were alternated throughout the experiment. The images were subsequently reconstructed to high resolution using the TRIGR algorithm (Chandra et al., 1996; Hanson et al., 1996). FAIR images were produced by subtracting flow-insensitive from flow-sensitive images. In order to determine if cerebral blood flow was altered in exercise versus sedentary animals under hypercapnic and normocapnic stimulation, region of interest (ROI) analysis was performed on all images. ROIs of 1 mm were chosen within the forelimb region of MC and the striatum of all animals. Intensities of each cortical ROI were normalized to those of the striatum of each animal in the manner described in experiment 1. Since no significant asymmetries between hemispheres were observed, the values were combined to produce the average normalized signal intensity for both hemispheres.

Experiment 3 Subjects. Forty-five female Long-Evans hooded rats (Rattus norvegicus) participated in this experiment. All animals were approximately 5 months old. Prior to the onset of the experiment, the animals were housed together in pairs in a standard laboratory cage and maintained under a 12-h light/dark schedule. All rats were allowed ad libitum access to food and water. Training. Each rat was assigned to either a VX (running wheel; circumference⫽111.8 cm) or IC (standard laboratory cage) group. Distance traveled by the VX animals was measured each day. Groups of five rats from the VX condition were killed after 1, 3, 5, 7, 10, 20, and 30 days of exercise in order to determine not only if exercise induced angiogenesis but also the developmental time course of this process. Groups of IC animals were killed after 5 and 30 days of inactivity in their home cages. All animals were killed via an i.p. injection of sodium pentobarbital (1 ml; 65 mg/kg). The rats were then perfused intracardially with 0.9% heparinized saline followed by 4% paraformaldehyde in 0.1-M PB. The brains were removed, postfixed for 24 h in 4% paraformaldehyde and then cryoprotected for 3 days in 30% sucrose in 0.1-M PB. Following cryoprotection, the brains were blocked and MC (from 1.2 mm anterior to 1.30 mm posterior to bregma) and visual cortex (occipital cortex, area 2, mediomedial [OC2MM] and occipital cortex, area 2, mediolateral) (from 3.80 mm to 8.3 mm posterior to bregma) were sectioned at 52 ␮m on a freezing microtome. Immunohistochemistry procedure. Following sectioning, the tissue sections were washed in PBS and then soaked in 0.3% hydrogen peroxide for 60 min at room temperature to inactivate the endogenous peroxidases. The tissue was then washed in 2% horse serum (HS) in PBS before being placed in a blocking solution (10% HS and 0.5% Triton-X in PBS) overnight at 4 °C. On the second day, the sections were washed first in PBS and then 2% HS in PBS followed by incubation in the primary antibody (F11; Pharmingen; 1:100 dilution in 1% HS, 0.5% Triton-X in PBS) overnight at 4 °C. On the third day, the sections were washed (2% HS in PBS) and transferred to a secondary antibody solution (10%

HS and 0.5% biotinylated, horse anti-mouse IgG secondary antibody in PBS; Vector Laboratories) for 90 min at room temperature. The tissue was then washed twice in 10% HS and 0.5% Triton-X in PBS before incubation in ABC (Vector Laboratories) for 60 min at room temperature. The sections were then washed in PBS followed by Tris buffer. Sections were then transferred to a DAB solution (100 mg DAB, 1.39 g nickel ammonium sulfate and 10 ␮l hydrogen peroxide in 200 ml Tris buffer) for approximately 10 min. Sections were then washed four times in PBS, mounted on gelled slides, and allowed to dry overnight before counterstaining with pyronin Y. For each animal, one section was used as an immunohistochemical control, which included the following: 1) incubation with buffer excluding the primary antibody, 2) incubation in the ABC and, 3) incubation in DAB. No labeling was found on these slices. Immunolabeling quantification. All evaluations of immunolabeling and hence angiogenesis were conducted with the analyst blind to the experimental conditions. Four immunolabeled sections from both motor and visual cortex were magnified (40⫻). Camera lucida drawings of immunostained blood vessels were then obtained from layers II/III and layer V. A 1-cm⫻1-cm point grid was then superimposed on the drawings and the number of labeled vessel intersections with the points on the grid as well as the total number of points falling within the drawing sample were determined. Using the stereological procedures described by Gundersen and coworkers (1988), the ratio of the number of points falling upon blood vessels to the total number of points in the sample area constitutes an unbiased estimate of the volume fraction of the cortex that is occupied by actively growing capillaries (i.e. the volume of actively growing capillary per unit volume of tissue).

RESULTS Experiment 1 For part one of our experiment, the only substantive difference between our two groups of animals was that VX rats ran from 158 to 10,091 (median⫽1,247) rotations per 24-h period. IC animals remained relatively inactive. Fig. 1 shows representative spin- and gradient-echo images from both VX and IC animals (approximately 0.20 mm anterior to bregma). Image analyses were conducted off-line using NIH Image 1.57 and were performed with the analyst blind to the experimental conditions. The average signal intensity of individual pixels in a 1-mm ellipsoid was calculated in four regions of noise (N), the MC in both hemispheres and background (BG) regions of brain in both hemispheres, for which we chose striatum, the mean signal intensity of which did not differ between groups [F(1,16)⫽1.62, P⬎0.20]. We chose striatum as our control area because 1) signal intensity in this region did not differ between the groups, 2) it is prominent within our sections (white-matter regions such as the anterior commissure are small in this region and offer limited sampling area), and 3) experimentally, the striatum is associated with slower movements than those in which our animals were engaging (Graybiel, 1990). Fig. 2 shows the regions sampled on a typical image. Because there were no significant asymmetries in signal intensity between hemispheres, the values were combined to yield the average signal intensity of both hemispheres. The signal-to-noise ratio (SNR) was thus derived for motor cortices using (⌺MC/2)/(⌺N/ 4)⫽SNRMC. BG activity was derived similarly [(⌺BG/2)/

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Fig. 1. Spin- and gradient-echo images. Representative spin- and gradient-echo images from a voluntary exercise (VX) rat (top panel) and its inactive control (IC) littermate (bottom panel). Spin-echo images (left) were used to localize slices to the same neuroanatomical plane. The loss of signal in the motor cortex of the gradient-echo (T-two-star-weighted) images (right) is consistent with an exercise-induced increase in capillary perfusion.

(⌺N/4)⫽SNRBG)]. The percent change in MC signal intensity from BG levels was then calculated using (SNRMC⫺SNRBG/SNRBG)⫻100⫽percent change in signal intensity. This calculation indicated that the proton signal intensity in VX MC was 30% lower than BG levels of brain compared with 11% for the IC rats. A one-way ANOVA with group as the between-measures factor indicated that this decrease was statistically significant [F(1,16)⫽5.31, P⬍0.035]. Analysis of signal intensity from muscle immediately adjacent to the brain revealed no systematic group effects [F(1,16)⫽1.02, P⬎0.33] nor did samples taken from non-motor regions of frontoparietal cortex [F(1,16)⫽.70, P⬎0.40] or from the medial septal nucleus [F(1,16)⫽.67, P⬎0.40]. Part two of this experiment indicated that the normalized OD of ferritin labeling was higher in the hilus of the dentate gyrus (ratio⫽2.51) than in the MC [ratio⫽1.07) F(1,12)⫽63.94, P⬍0.0002]. No significant group or group⫻structure differences were observed [F(1,12)⫽ 0.23, P⬎0.63 and F(1,12)⫽0.15, P⬎0.70 respectively]. As for the iron stain, normalized OD measurements indicated that labeling was more intense in the molecular layer of the dentate gyrus (ratio⫽2.89) than in the MC (ratio⫽1.54) [F(1,9)⫽23.82, P⬍0.0009]. No significant

group or group⫻structure differences were observed [F(1,9)⫽0.00, P⬎0.99 and F(1,9)⫽0.38, P⬎0.55 respectively]. Taken together, these findings indicate that the effects of exercise on signal intensity do not reflect differences in ferritin or iron levels between groups. Experiment 2 The rats in this experiment performed similarly to those of experiment 1, completing an average of 5673 rotations per day. Fig. 3 shows representative normocapnic and hypercapnic inversion recovery spin echo images from both VX and IC animals (approximately 0.20 mm anterior to bregma). At basal CO2 levels, the MC normalized to striatum showed no consistent differences between groups of animals [F(1,6)⫽0.04, P⬎0.85]. Under hypercapnia, though, the MC ROI signal intensity was 22% greater in VX rats than in ICs, suggesting that blood flow in response to CO2 was greater in these animals. A one-way ANOVA with group as the dependent measure indicated that this effect was significant [F(1,6)⫽11.06, P⬍0.016]. This differential response to CO2 was observed in all animal pairs examined.

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Fig. 2. Exercise-induced changes in signal intensity. (A) The average signal intensity of individual pixels in a 1-mm2 ellipsoid was calculated in four regions of noise (white ellipsoids located in four corners), the motor cortex (upper two ellipsoids in brain) in both hemispheres and background regions of brain (lower two ellipsoids in brain) in both hemispheres. The signal-to-noise ratio (SNR) was derived for motor cortices using (⌺motor cortex [MC]/2)/(⌺noise [N]/4)⫽SNRMC. Background (BG) activity was derived similarly (⌺BG/2)/(⌺N/4)⫽SNRBG. The percent change in MC signal intensity from BG levels was then calculated using (SNRMC⫺SNRBG/SNRBG)⫻100⫽percent change in signal intensity. (B) The percent change in MC signal intensity from BG levels indicated that the proton signal intensity of VX MC was 30% lower than BG levels of brain compared with 11% for the inactive control (IC) rats [F(1,16)⫽5.31, P⬍0.035].

Experiment 3 Calculation of the mean number of wheel rotations per night per group yielded the following averages for the VX1, VX3, VX5, VX7, VX10, VX20, and VX30 conditions respectively: 463, 1342, 1501, 3877, 1265, 5877, and 4909. Fig. 4 depicts the actively growing capillary-volume fraction for layers II/III and V of primary MC across respective days of training. As the graph indicates, some capillary labeling by the F11 antibody was present at each of the time points sampled in our experiment including the control or 0 day (C5 and C30). Exercise on the running wheels yielded a small increase in labeled tissue on day 3 followed by substantially more robust labeling on day 30. One-way ANOVA by layer with day (0 [C5 and C30], 1, 3, 5, 7, 10, 20, and 30) as the between factor confirmed that capillaryvolume fraction of layer II/III increased with exercise [F(7,34)⫽2.31, P⬍0.05]. Post hoc comparisons using the least significant difference (a modified form of t-test in which the error term is garnered from the omnibus F test) procedure indicated that the increase observed at day 30 was significantly elevated compared with all other time points (P⬍0.05 for all comparisons) with the exception of day 3. The small increase observed on day 3 did not differ

Fig. 3. Flow alternating inversion recovery images. (A) Representative inversion recovery spin echo (IRSE) (left), normocapnic (center), and hypercapnic (right) images from both inactive control (IC) (top) and voluntary exercise (VX) (bottom) animals. (B) Under hypercapnia, the motor cortex region of interest signal intensity was 22% greater in VX rats than in ICs, suggesting that blood flow in response to CO2 was larger in these animals [F(1,6)⫽11.06, P⬍0.016].

significantly from any other time point (P⬎0.05 for all comparisons). The main effect for layer V was not significant [F(7,34)⫽2.01, P⬍0.085]. Comparisons within this layer were similar to those reported for layer II/III (i.e. capillary volume fraction on day 30 was elevated compared with all time points except that of day 3). Analyses of visual cortex labeling were conducted only on tissue from inactive controls (C5 and C30 groups combined) and the 30-day VX animals. A one-way ANOVA of

Fig. 4. CD61 immunolabeling. Significant angiogenesis as indicated by F11 immunostaining of the ␤3 subunit of the ␣v␤3 (CD61) integrin was observed in layer II/III of motor cortex of voluntary exercise rats at 30 days following the onset of running (P⬍0.05).

R. A. Swain et al. / Neuroscience 117 (2003) 1037–1046

OC2MM capillary-volume fraction with group (C5⫹C30 and VX30) as the independent variable did not reveal a significant main effect [F(1,12)⫽3.64, P⬎0.08]. Similarly, a one-way ANOVA of data from area OCML also revealed no significant main effect for group [F(1,12)⫽3.14, P⬎0.10].

DISCUSSION The results of experiment 1 indicate that prolonged elevation of motor activity and accompanying activation of MC decrease proton signal intensity as measured by fMRI. Because proton signal intensity is inversely related to deoxyhemoglobin levels, a decrease in signal intensity is consistent with increases in blood perfusion. A perfusion signal change could arise from increased numbers of capillaries within MC, relaxation of arterioles (alterations in blood flow) within MC, a change in the size or number of extracerebral vessels overlying MC, or an alteration of tissue oxygen utilization. Of course, these phenomena need not be mutually exclusive. It is unlikely, though, that the changes in signal intensity were due to alteration of extracerebral vessels supplying the facial and head musculature as our analyses found no differences between groups. Differentiation between blood flow and capillary angiogenesis was not possible with our imaging parameters. T2*-weighted imaging is sensitive to deoxyhemoglobin concentration per unit brain tissue, which would be altered under both circumstances. If our results were due to a chronic increase in blood flow as a result of arteriole relaxation, then this flow must be accompanied by greater oxygen utilization as well. Greater blood flow without a concomitant increase in oxygen utilization would yield an increase in parenchymal signal intensity (the opposite of our findings) because the overall ratio of oxygenated hemoglobin (non-paramagnetic) to the deoxygenated form (paramagnetic) would increase. Angiogenesis, by altering the density of blood vessels within an unchanging neuropil volume (and hence the level of total deoxyhemoglobin), would decrease the proton signal intensity with or without a concomitant increase in oxygen utilization. It is conceivable that the loss of signal intensity in the T2*-weighted images collected in this study could result from some other physiological mechanism apart from angiogenesis. For example, ferritin, while heterogeneously distributed in the brain, can be localized to perivascular cells, primarily microglia and oligodendroglia (Connor and Benovic, 1992; Connor et al., 1994; Connor and Menzies, 1995) and it has been suggested that it may subserve image contrast in MRI (Vymazal et al., 1996). Our analysis of ferritin incorporation in the MC of rats trained similarly to those in the fMRI portion of the experiment failed to find any significant group effects. Furthermore, a review of the literature failed to locate any reports of exercise-induced increases in ferritin/iron incorporation. In fact, our search found considerable evidence that iron and serum ferritin levels are likely to be depleted as a result of prolonged exercise (Newhouse and Clement, 1988). We suggest,

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therefore, that the present results may serve as the first documentation of a T2* effect consistent with a chronic increase in vascular perfusion. The FAIR results for rats under normocapnia are consistent with the results of the T2*-weighted imaging insofar as an increase in blood volume without any flow increase would result in a signal deficit on T2*-weighted images. An increase in flow would deliver increased levels of arterial, oxygenated blood to the area and would tend to counter the effects of a blood-volume increase. Several interpretations of the FAIR results under hypercapnia are possible. The simplest and perhaps most parsimonious is that the capillaries of our exercised animals respond more exuberantly to activation by elevated CO2. Blood flow in our VX animals was nearly 28% larger than that observed in our ICs. Vessel dilation and hence blood-flow increases in response to hypercapnia have been documented extensively (Abounader et al., 1995; Atkinson et al., 1990; Bereczki et al., 1993; Duelli and Kuschinsky, 1993; Hudetz et al., 1997; Shockley and LaManna, 1988). It might be proposed that the large increase in flow that we observed is due to a “practice effect.” Certainly, our animals had placed considerable demands on the vasculature of the MC to meet the oxygen and glucose needs of neurons exposed to 30 days of strenuous exercise. If it is true that capillary dilation becomes more robust with experience, then this phenomenon would serve as one more indicator of the plastic nature of the cerebral vascular supply. Our hypercapnia results may also be due to changes in the variability of blood flow. Adjacent cerebral capillaries may display distinctly different flow velocities (Abounader et al., 1995; Vogel et al., 1997). In fact, plasma and red blood cell velocities may differ in a single capillary (Hudetz et al., 1993; Rovainen et al., 1993). Some investigators (Villringer et al., 1989, 1994; Pawlik et al., 1981; Tajima et al., 1992) have even suggested that approximately 10%– 15% of the capillaries in the brain may be perfused only by plasma (no red blood cells). Under hypercapnia, these “plasmatic” vessels as well as normal vessels would increase red blood cell flow and thus would reduce the variability associated with cerebral blood flow during normocapnia (Abounader et al., 1995; Vogel et al., 1997; Hudetz, 1997; Hudetz et al., 1997). For this mechanism to underlie our results, capillary blood flow in VX MC would have to display less variability than vessels in the IC animals or would have to occur in a larger pool of vessels, a proposition that we support in experiment 3. One final possibility is that the increased signal intensity observed in the MC of exercised rats is congruent with the recruitment of previously nonperfused capillaries from a brain capillary reserve or from a new pool of nonperfused vessels, the growth of which was induced by exercise. It has been proposed by some investigators that mammals have at least some capillaries in the brain apportioned into a cerebrovascular reserve that is not normally perfused under resting conditions (Weiss et al., 1982; Weiss, 1988). Portions of the capillary reserve open in response to activation of neighboring neural tissue (Bruhn et al., 1994) or

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in response to hypercapnia (Frankel et al., 1989; Weiss et al., 1982). The presence or absence of a cerebral capillary reserve has long been a controversial issue (Petren, 1938, Diamond et al., 1964; Weiss et al., 1982) and one that our data can neither verify nor disprove. The results of experiment 3 provide definitive evidence that repetitive exercise promotes angiogenesis in the MC of the adult mammalian brain. In addition, these data suggest that running-dependent capillary growth is restricted to or greatest in motor regions of the brain at 30 days. Our results plus those of Black and colleagues (1990), who demonstrated blood-vessel addition in the cerebellum following 30 days of activity on running wheels, suggest that capillary plasticity is a general feature of motor systems of the brain in response to exercise and concomitantly elevated neural activity. In fact, prolonged periods of neural activation may support capillary plasticity in any cortical region of the brain in a task-specific manner. For example, rats reared in visually rich ECs display not only increases in visual cortex dendritic arbor and synapse number but also significant increases in capillary volume within the same region of cortex (Sirevaag et al., 1988). Whether subcortical regions display similar forms of capillary plasticity remains an open question. It is known, however, that subcortical regions such as the hippocampus are responsive to exercise. Cotman and colleagues (Neeper et al., 1996) have reported that levels of brainderived neurotrophic factor (BDNF), a neurotrophin, increase substantially in the rat hippocampus following 2, 4, or 7 days of running-wheel activity. In a subsequent experiment, this group has reported that mRNA for BDNF increases within 6 h following onset of running on a wheel (Oliff et al., 1998). Recently, van Praag et al. (1999) have examined neurogenesis in the dentate gyrus of the adult rat and found that exercise on a running wheel results in a near doubling of the number of newly born neurons that survive. Whether the changes reported above are accompanied by angiogenesis is unknown. General conclusion This study adds to the small but growing literature reporting angiogenesis in the adult brain in the absence of pathology such as tumors (Opitz, 1951; Ausprunk and Folkman, 1977; Knighton et al., 1983; Black et al., 1990). While interpretation of an early report of exercise-induced angiogenesis in MC was complicated by severe stress (Petren, 1938), a number of reports of cerebral cortical angiogenesis in adult rats placed in ECs (Black et al., 1991) and in rats exposed to hypoxia (Harik et al., 1995) argue against previous reports that cortical angiogenesis is complete by 21 days of age (Gyllensten, 1959; Caley and Maxwell, 1970; Bar, 1980; Rowan and Maxwell, 1981). The present results do suggest, however, that running exercise-induced plasticity of vascular perfusion and angiogenesis in the forebrain is restricted to or greatest in motor regions of the cerebral cortex activated by the task. Neither frontoparietal cortex nor subcortical structures displayed any such changes.

The enhanced perfusion of MC documented here also complements recent findings of MC synaptogenesis following motor skill acquisition but not repetitive motor activity (Kleim et al., 1996). Together, these studies parallel synaptic and vascular plasticity described in cerebellar cortex (Black et al., 1990) and suggest a distributed pattern of CNS adaptation in fore- and hindbrain to behavioral demands, with learning causing synaptogenesis and activity causing angiogenesis. Finally, these results demonstrate that fMRI may be a viable noninvasive tool for imaging chronic changes in brain perfusion or oxygen utilization that should be useful in examining effects of exercise and other prolonged experience manipulations in humans. This technique also promises noninvasive assessment of other clinical interventions such as rehabilitation therapy following brain injury. Acknowledgements—Supported by grants from the Retirement Research Foundation and NIH AG 10154 to W.T.G. and the NIH Research Resource (PHS 5 P41 RR05964) and Servants United Foundation to P.C.L.

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(Accepted 20 September 2002)