(Stroke. 1995;26:2135-2144.)
© 1995 American Heart Association, Inc.
Articles |
Neocortical Neural Sprouting, Synaptogenesis, and Behavioral Recovery After Neocortical Infarction in Rats
From the School of Biological Sciences, Division of Neuroscience, University of Manchester (England) (R.P.S.), and the Departments of Neurology (T.A.K.) and Anatomy and Neurosciences and Marine Biomedical Institute (C.E.H.), University of Texas Medical Branch, Galveston.
Correspondence to Dr C.E. Hulsebosch, Department of Anatomy and Neurosciences, 301 University Blvd, 1069, University of Texas Medical Branch, Galveston, TX 77555-1069.
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Background and Purpose Neuroanatomical plasticity is well described in lesions of the hippocampus but remains a subject of some controversy in the neocortex. The purpose of the present study was to measure the neocortical distribution and density of expression of proteins known to be involved in neurite growth or synaptogenesis and to correlate the neocortical expression with behavioral recovery after a focal neocortical infarction. Focal neocortical infarction creates a circumscribed lesion in the neocortex that provides a denervation stimulus for neurite growth and synaptogenesis.
Methods Unilateral neocortical ischemia was induced in male spontaneously hypertensive Wistar rats (n=4 per time point) by permanent occlusion of the distal middle cerebral artery and ipsilateral common carotid artery. To determine the spatial and temporal distribution of neurite growth and/or synaptogenesis, GAP-43, a growth-associated protein expressed on axonal growth cones, and synaptophysin, a calcium-binding protein found on synaptic vesicles, were examined by immunohistochemical techniques. The reaction product was measured, and the distribution was recorded. Since the resulting infarction included a portion of the forelimb neocortex, behavioral assessments of forelimb function that used the foot-fault test of Hernandez and Schallert were performed on the same rats used for immunohistochemical studies. Recovery times were 3, 7, 14, 30, and 60 days after surgery.
Results Both GAP-43 and synaptophysin proteins demonstrated statistically significant increases in the density of immunoreaction product as determined by optical density measurements in the neocortex of infarcted rats compared with sham controls. The GAP-43 was elevated to statistically significant levels in forelimb, hindlimb, and parietal neocortical regions medial and lateral to the infarction only at days 3, 7, and 14. In contrast, synaptophysin demonstrated no statistically significant changes in expression at 3 or 7 days but demonstrated statistically significant increases at 14, 30, and 60 days in the forelimb, hindlimb, and parietal neocortical regions medial and lateral to the infarction as well as in the contralateral parietal neocortex. Behavioral assessment of forelimb function indicated impairment of forelimb placement on the side contralateral to the infarction that trended toward control values at 14 days and was not significantly different from controls by 30 days.
Conclusions These data support the occurrence of neurite growth followed by synaptogenesis in the neocortex, ipsilateral and contralateral to neocortical ischemia, in a pattern that corresponds both spatially and temporally with behavioral recovery. Thus, neuroanatomical remodeling in the neocortex provides a mechanism for recovery of function.
Key Words: cerebral ischemia • immunohistochemistry • neuronal plasticity • synaptophysin • rats
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The existence of central nervous system (CNS) plasticity after trauma is a subject of some controversy.1 2 3 4 In at least one CNS region, the hippocampus, extensive studies have provided evidence that plasticity does occur after various ablation models.1 2 This plasticity establishes the restoration of function by changes in electrophysiological, anatomic, and biochemical parameters. Consequently, it is reasonable to predict that similar events occur after trauma in other regions of the CNS. In the period after cerebral ischemic infarction, massive neuronal death will result in regions of denervation, which could provide a stimulus for undamaged neurons to sprout and establish new synaptic connections. This sprouting and synaptogenesis would support the hypothesis of neuronal remodeling and plasticity after cerebral infarction. Axonal and dendritic sprouting and alterations in the number of synapses will be accompanied by an increase in proteins involved with neurite sprouting and in the synaptic vesicle population in this region. These processes and their long-term effects have not been well researched in the cerebral cortex after cerebral ischemia.
Axonal sprouting, a component of anatomic plasticity, can be identified by the elevated expression of GAP-43, a growth-associated protein with a molecular weight of 43 kD.5 GAP-43 is a membrane-bound protein found in the axonal growth cones of sprouting CNS axons.5 6 7 8 9 10 11 12 13 Another protein useful in the identification of axonal sprouting and synaptogenesis is synaptophysin, a presynaptic vesicle protein (Mr, 38 000) that is found in virtually all nerve terminals. It is believed that synaptophysin forms homo-oligomers, probably a hexamer.14 Levels of synaptophysin within the terminal are believed to remain constant along with several other vesicle proteins as a result of the recycling of vesicle material in the nerve terminal.15 16 17 Synaptophysin has been used by a variety of laboratories to quantitate numbers of terminals during neuroanatomical remodeling and neural development.18 19 20 21 22 23 24
It is the objective of this study to use quantitative immunohistochemical techniques to determine increased expression by density and distribution measurements of GAP-43, as an indicator of axonal sprouting, and of synaptophysin, as an indicator of the number of synaptic terminals. The expression of these proteins will be measured in areas of the neocortex after distal middle cerebral artery (MCA) occlusion with the use of the unilateral tandem occlusion model of Chen et al25 and Brint et al,26 in which focal cerebral cortical ischemia is produced by permanent distal MCA occlusion in spontaneously hypertensive Wistar rats (SHR). The use of the SHR strain provides spatially consistent and large neocortical infarct volumes because of the lack of anatomic variation in the MCA and the limited collateral circulation from anterior and posterior cerebral arteries. This results in highly reproducible, well-circumscribed focal ischemic injuries with very little to no penumbral region and no ischemic damage to subcortical structures.25 26 27 Thus, interanimal variability is reduced. Reproducibility of this focal ischemia model provides a baseline for assessing changes in gene expression and behavioral recovery.
Since an important outcome measure is recovery of function, it is of
interest to correlate the expression of GAP-43 and synaptophysin with
functional outcome. The region of neocortical ischemia produced
in this model includes a portion of the forelimb neocortex (Fig 1
). Consequently, forelimb dysfunction on
the side contralateral to the ischemia is predicted. If the
temporal expression of the proteins involved in neuroanatomical
remodeling in appropriate neocortical regions (ie, the forelimb)
corresponds to the improvement of forelimb function, then these data
would provide support that neuroanatomical remodeling provides a
mechanism for recovery of function in this model. Consequently, another
aim of this study is to test forelimb function over time and compare
the temporal behavioral data to the temporal pattern of protein
expression.
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Surgical Methods
Nonfasted male SHR (weight, 260 to 300 g) were anesthetized with halothane (4% induction/1% maintenance), placed on a heating pad, and given an antibiotic (streptomycin, 0.10 mL, 150 mg/mL IP). SHR were used to ensure constant infarction volume and placement because of poor collateral circulation and consistent vascular anatomy.26 The right common carotid sheath was exposed by a ventral midline incision, the sheath was removed, the vagus nerve was separated, and the common carotid was permanently ligated with 4-0 suture. The rat was placed in a head clamp, an incision was made between the right eye and ear, and the underlying temporalis muscle was excised. A saline drip was started on the exposed skull, and a craniotomy was performed 1 to 2 mm rostral to the squamosal fusion. The underlying dura was penetrated, and the MCA was permanently ligated with a 10-0 suture knot tied proximal to the frontal branch. The wounds were then sutured shut with 4-0 Prolene, and the animal was taken off anesthesia and allowed to recover in a cage on a heating pad for 24 hours. Sham controls were prepared for surgery as described with the exception that the 10-0 suture was not tied into a knot but was left in place. The animals recovered within 2 hours after surgery was completed. If an animal survived 12 hours after surgery there was no subsequent mortality. Functional damage could not be detected by gross motor and sensory observations. Antibiotics (streptomycin, 0.10 mL, 150 mg/mL per day IP) were given for 2 days after surgery. The animals were then housed two to a cage. All surgical procedures were approved by the University of Texas Medical Branch Animal Care and Use Committee.
Immunohistochemistry Methods
At various time points after surgery (3, 7, 14, 30, and 60
days), the animals (n=8 for each time point, of which 4 per time point
were sham control animals) were anesthetized with sodium
pentobarbital and perfused transcardially with 4%
paraformaldehyde fixative in phosphate buffer
containing 0.1% picric acid. Brains were removed, photographed, taken
through graded sucrose solutions in fixative up to 30%, blocked,
embedded in OCT freezing compound (Miles, Inc), frozen, and stored at
-70°C. Cryosections were sectioned at a thickness of 60 µm. The
sections were run as sets, with sections from infarcted brains and from
sham control brains run simultaneously for a single primary
antibody in partitioned devices with screened bottoms that were dipped
in polymerized methyl methacrylate boxes filled with the appropriate
solutions. Thus, all sections were exposed to the same solutions. The
free-floating sections were immunostained with the use
of a monoclonal antibody to either GAP-43 (Boehringer Mannheim)
or synaptophysin (Boehringer Mannheim) at a dilution of 1:500.
Every fifth section was mounted on gelatin-coated slides for silver
staining.29 To ensure that immunostaining
was specific, control reactions were immunostained in the
same solutions with no primary antibody, with glial fibrillary acidic
protein primary antibody (1:500, Boehringer Mannheim), or with
a leukocyte common antigen primary antibody (1:500, Chemicon). The
tissue processed in the absence of primary antibody had little
immunostaining. The glial fibrillary acidic protein
antibody and the leukocyte common antigen primary antibody caused
immunostaining that was specific for astrocytes or
leukocytes, respectively, with relatively homogeneous
staining with the exception of a narrow band of immunoreaction
surrounding the infarction, which was subtracted from the region
analyzed for GAP-43 and synaptophysin. The immunoreaction was
visualized with the use of standard avidin-biotin horseradish
peroxidase/diaminobenzidine techniques (Vectastain Elite ABC kit), and
sections were mounted on gelatin-coated microscope slides (1%
gelatin, 0.1% potassium chromium sulfate).
Optical density measurements of immunoreacted tissue were made from an enlarged image (x500) with the use of a QX-7 Image Analyzer System (Quantex Corp). With the area operation, a standard square was generated that was equivalent to 0.2x0.2 mm of tissue (actual size). The area function is preferable to point measurements since any given region can vary by 19 radiance levels without appearing to have variation to the eye. The square sample area was used to measure the mean radiance (light transmission) on a scale of 0 to 256 for five repeated measures within an anatomic region (medial and lateral in the neocortex adjacent to the ischemic damage) in which the density of the reaction was within the range determined to be signal by threshold methods described below. Limits of ischemic damage were determined in adjacent ammoniacal silver–stained sections. Readings were made in a line perpendicular to a tangent of the surface of the cortex, restricting the multiple readings within accepted cortical regions.30 For comparison, similar measurements were taken from analogous locations in similar anatomic regions of similar sections (with respect to bregma and interaural coordinates) from sham control sections that were processed for immunoreactivity at the same time as the experimental sections. Background levels of immunoreactivity for each section were determined by measuring areas of white matter such as the corpus callosum, internal capsule, and anterior commissure. These structures were chosen because GAP-43 and synaptophysin levels are low in these regions as a result of an absence of axon terminals. These background levels (mean, 186±33) were subtracted from the cortical measurements and normalized to a scale of 0 to 100, where 0 equals 0% blockage of transmitted light (optically transparent) and 100 equals 100% blockage of transmitted light (optically opaque), allowing the establishment of percent optical densities. Optical densities on the ischemic sides, medial and lateral to the injury, and the contralateral nonischemic sides were then compared with optical density measurements from similar regions in similar sections from sham controls. The optical density values were normalized such that the sham control optical densities were normalized to 1 for each time point to allow comparisons between groups. With QX-7 software, it is possible to determine the threshold for the density of reaction by adjusting gray scale assigned colors with the LUT (look-up table) function of the menu. This technique allows easy visualization of reaction product that is determined to be signal because of the density of the reaction product. After adjusting black levels to standardize background measurements in regions empirically determined as background in the white matter (mean background measure, 186±33), we empirically determined that the signal was radiance readings in the range of 0 to 94 on a scale of 256 (0 is optically opaque, and 256 is optically transparent). The look-up table was altered to assign all values from 0 to 94 a red color, which is easy to visualize and standardizes the regions to be selected for both optical density readings and area measurements. We used this method to calculate the areal distribution of immunoreaction of GAP-43 at 3 days in the cortical regions medial and lateral to the infarcted region and of synaptophysin at 60 days in the cortical regions medial and lateral to the infarcted region and on the contralateral cortex. These two time points were selected because the expressions of the two protein species were determined empirically to be optimal at these time points. Both radiance and areal measurements were made in regions determined to be signal from histological sections, and the areal readings were calibrated with the LOATS software calibration function into millimeter readings. Areal measurements of the infarct region were performed with the use of a grid placed on printed enlargements (x8) of photographs taken of the infarcted side of whole brains. Infarct volumes were calculated from the areal measurements multiplied by the neocortical depth based on the mean cortical depth measured in histological sections from similar cortical regions in sham control sections. The slides were coded and the data collected by laboratory personnel unaware of the surgical status or time from injury to remove investigator biases. The data were analyzed with the use of ANOVA for multiple comparisons and Student's t test for comparison between two groups (such as experimental groups compared with preoperative or sham control values), with P<.05 set as the level of confidence.
Behavioral Methods
For the behavioral analysis, the rats were placed on
elevated hexagonal grids of two sizes to test placement dysfunction of
the forepaw with the use of the Hernandez-Schallert foot-fault
test.31 The grids had openings of either 3 cm (small) or 6
cm (large). Rats place their paws on the wire while moving along the
grid. The rats were videorecorded from below the grid for ease
in recording the stepping pattern. With each weight-bearing
step, the paw may fall or slip between the wires. This is recorded
as a foot fault. The number of faults for the forepaw contralateral to
the infarction is recorded along with the number of successful
steps and displayed as a percentage of contralateral forelimb foot
faults per forelimb steps. Faults were calculated for both sizes of
grids. Baseline percentages were acquired by testing sham-operated
rats and rats preoperatively (control). These faults are usually
symmetrical and occur less than 10% of the time (see Fig 9). To reduce olfactory cued behavior during grid
testing, the apparatuses were cleaned with a disinfectant
(Quinticare) between tests. The data were analyzed with the use
of ANOVA for multiple comparisons and Student's t test for
comparison between two groups (such as experimental groups compared
with preoperative or sham control values), with P<.05 set
as the level of confidence. Correlations of immunohistochemical results
and recovery on behavioral testing were analyzed for
significance with a three-way ANOVA test for comparison between
groups of animals, with P<.05 chosen as the level of
confidence.
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At the time the animals were killed, the region supplied by the occluded MCA, principally the parietal cortex, appeared infarcted as determined by gross inspection because of a lack of color from the picric acid in the perfusate and an inconsistent texture compared with the surrounding cortex. The average surface area of the infarction in 10 brains chosen at random was 58.4±2.8 mm2. The mean volume of the infarction was calculated to be 86.4±8.0 mm3 and was confined to the neocortex (Fig 2
). By comparison, the contralateral side
appeared undamaged by both gross inspection and in
histological sections. It should be noted that the
histology of the hippocampus and other subcortical structures appeared
normal in hematoxylin and eosin–stained sections, in
thionin-stained sections, and in ammoniacal silver–stained
sections. Areal cell counts of hematoxylin and eosin–stained
sections of the hippocampal CA1 region ipsilateral and contralateral to
the infarcted side demonstrated no difference in pyramidal
cell number between sham controls and infarcted animals or between
sides in the infarcted animals. Both of the lateral ventricles and the
third ventricle appeared enlarged relative to noninfarcted animals, as
reported earlier.32 33 For the immunoreaction product,
it should be noted that intensity of reaction product varied with
each reaction and from animal to animal but was consistent
within each animal and within each section. In all comparisons, there
was no statistical difference between preoperative and sham control
values. The level of confidence displayed is that calculated between
the experimental group compared with the sham control group as
determined by Student's t test, except where noted.
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GAP-43–like immunoreactivity was demonstrated in the cortex of both
hemispheres and in the hypothalamus with little immunoreactivity
demonstrated in the white matter. In general, the GAP-43–like
immunoreactivity was diffuse within gray matter structures (Figs 3 and 4). The optical density measurements of
the cortex medial and lateral to the area of infarction and the
analogous cortex in the contralateral hemisphere are graphed in Fig 5. At 3, 7, and 14 days after infarction, the reaction
product had statistically significant higher optical densities
compared with similar cortical regions from sham controls
(P<.05). GAP-43 immunostaining was also
elevated at these time points in the cingulate and entorhinal cortices
of both hemispheres and in the thalamus in some animals. At 30 and 60
days after infarction, there was no difference in staining between the
two hemispheres compared with sham controls. Elevated staining was also
present in the hippocampus, dentate gyrus, and septal/hypothalamic
regions in sections of sham controls and at all time points after
occlusion. The areal measurements of the GAP-43 density in sections at
bregma -0.30 and interaural 8.70 were 0.99±0.34 mm2
medial to the infarct in the hindlimb neocortical region and 1.77±0.13
mm2 lateral to the infarct in the parietal 2 neocortical
region. The areal measurements of the GAP-43 density in sections at
bregma 0.70 and interaural 9.70 were 2.17±0.84 mm2,
which was just anterior to the region of the infarction in the forelimb
neocortical region.
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Synaptophysin-like immunoreactivity was demonstrated in the
neocortex and hippocampus of both hemispheres, with no immunoreactivity
demonstrated in the corpus callosum. Synaptophysin immunoreactivity was
also observed in the pyriform cortex. In general, the
synaptophysin-like immunoreactivity was diffuse within gray matter
structures (Figs 6 and 7). Shortly after
the infarction (3 and 7 days), there was no difference in
immunoreactivity between the two sides of the brain and sham controls.
At the later time points (14, 30, and 60 days), the reaction
product had a statistically significant (P<.05) higher
optical density in regions medial and lateral to the ischemic
neocortex, which correspond to the forelimb and hindlimb regions and
the parietal 1 and 2 regions, and in analogous regions in the
contralateral hemisphere, which correspond to parietal 1 and 2 regions,
compared with sham controls (Fig 8). The
areal measurements of the regions of synaptophysin densities 60 days
after occlusion in histological sections at bregma
-0.30 and interaural 8.70 were 1.30±0.51 mm2 medial to
the infarct in the hindlimb neocortical region and 1.44±0.40
mm2 lateral to the infarct in the parietal 2 neocortical
region and 5.03±0.79 mm2 in the contralateral neocortical
parietal 1 region. The areal measurement of the synaptophysin density
in sections at bregma 1.60 and interaural 10.60, which was just
anterior to the region of the infarction in the forelimb neocortical
region, was 0.81±0.71 mm2.
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The behavioral data indicate that the forelimb contralateral to the infarction demonstrated a statistically significant increase in forelimb foot faults compared with sham controls. The difference in performance between the infarcted animals and sham controls on the small grid was statistically significant at postoperative days 3, 7, 14, and 20 but not at 30 days (P<.0005, P<.0005, P<.01, P<.03, and P=.25, respectively). On the large grid, the percentage of foot faults was statistically significant at all time points tested (P<.00005, P<.0001, P<.00001, P<.00005, and P<.1x10-6, respectively).
There was a correlation of improved behavioral performance on the Hernandez-Schallert foot-fault test and elevated GAP-43 immunoreactivity between infarcted and sham control groups at 7 days that was significant on both grid sizes (P<.05). At 3 days after infarction, there was a trend toward significance in the correlation of improved performance and GAP-43 immunoreactivity between groups on the small grid in the Hernandez-Schallert foot-fault test. There was a statistically significant correlation of improved foot-fault behavior and elevated synaptophysin immunoreactivity between infarcted and sham control groups at all time points for the small grid performance, that is, the numbers of foot faults were at maximal values at the same time points that the synaptophysin optical densities were at minimum values, on postoperative days 3 and 7. In contrast, at 14, 20, and 30 days the numbers of foot faults were at minimal values, whereas synaptophysin optical densities were at maximum values in infarcted animals compared with sham control values. These correlations were statistically significant (P<.05). There was no correlation over time between the number of foot faults and elevated synaptophysin optical densities for the large grid performance.
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Two proteins involved with neurite growth—GAP-43 and synaptophysin—were examined in focal cortical infarction. That GAP-43 is involved with initiation of neurite growth is supported by a variety of experiments. GAP-43 inhibits some Gi proteins to regulate metabolic responses that initiate axonal growth.10 11 The importance of GAP-43 in this regulation has been demonstrated in cultured rat pheochromocytoma (PC-12) cells where neurite outgrowth is eliminated by blocking GAP-43 expression with anti-sense oligonucleotide probes.12 Conversely, the transfection of fibroblasts with the GAP-43 gene results in GAP-43 expression and the extension of neuritelike processes.13 GAP-43 is expressed at high levels during neuronal growth in early development but is normally found in lower levels in adults. Lesions of adult hippocampal pathways in which the result is known to be axonal sprouting demonstrate increases in the expression level of GAP-43, which then returns to basal levels once sprouting has finished.18 34
The increase of GAP-43 immunoreactivity after ischemia in the present study is interpreted as evidence of axonal growth and sprouting in the neocortical regions surrounding the infarction, which correspond to the forelimb, hindlimb, and the parietal 1 and 2 regions. This axonal sprouting is most robust within days after occlusion, then diminishes by 30 days. This suggests that denervation, with subsequent loss of afferent input and upregulation of neurotropins, promotes axonal sprouting. Sprouting will occur during a temporal window induced by a lesion stimulus and subsequently attenuates once synapses are formed and neurotropin levels presumably decrease. A few reports in the literature support the hypothesis of the ability of the cortical neurons to sprout after other deafferentation paradigms.35 36 37 38 For example, studies using the model of the removal of rat vibrissae demonstrated a slight increase in GAP-43 immunoreactivity in the contralateral barrel cortex within 1 week after surgery, which is interpreted to be from intrinsic cortical neurons.36 39 In situ hybridization experiments following ischemia in humans demonstrated increases in GAP-43 mRNA shortly after the ischemic lesion.35 However, other studies have demonstrated GAP-43 mRNA expression after lesions of the CNS without the concomitant expression of the GAP-43 protein,40 indicating the possibility of posttranslational degradation and the importance of GAP-43 protein expression if correlations are to be made with axonal sprouting.41 42 43
Synaptophysin, one of several vesicle proteins, remains constant within a terminal because of the recycling of vesicle material in the nerve terminal15 16 17 and has been used by a variety of laboratories to quantitate numbers of terminals during neuroanatomical remodeling and neural development.18 19 20 21 22 23 24 Transfection of immortalized epithelial cell lines to express synaptophysin results in the formation of synaptophysin-containing vesicles, with synaptophysin being the only major vesicle membrane protein.14 Additionally, these synaptophysin-containing vesicles have biophysical properties that are very similar to those of presynaptic vesicles.14 Methods developed by Masliah et al19 allow the estimation of increases or decreases in synaptic numbers with the use of synaptophysin immunostaining and are used by others in the fields of anatomical remodeling and neural development.18 19 20 21 22 23 24 The increase in synaptophysin-like immunoreactivity at 14 days after distal MCA occlusion supports the hypothesis that an increase in the number of synapses has occurred in the neocortical regions adjacent to the ischemic lesion, which correspond to the forelimb, the hindlimb, and the parietal 2 regions. In addition, the contralateral cortex demonstrated increased synaptophysin-like immunoreactivity in the analogous regions as well as parietal 1, which was destroyed ipsilaterally by the neocortical ischemia. The continued difference between the experimental group and the sham group suggests that the increase in synaptic numbers is permanent. This is in agreement with reports of an increase in dendritic branching seen by others after neural trauma.1 2 37 38 An increase in dendritic branching would allow for synaptogenesis by providing more surface area and thus greater potential for axodendritic connections for neural communication.
The sequence of elevated GAP-43 immunoreactivity after ischemic insult followed by increased synaptophysin immunoreactivity is temporally consistent with the hypothesis that there is axonal sprouting followed by synaptogenesis in the cortex surrounding an area of infarction and in the contralateral cortex. The denervation of the peri-infarct cortex and the contralateral cortex (from lost callosal projections) provide the stimulus to remodel. This plasticity is important in that a deafferented area of cortex will not lose its ability to integrate functionally with other regions of the cortex. Similarly, neurons that have lost efferent target neurons may integrate into new neuronal circuitry. The ability of neurons to demonstrate neuroanatomical remodeling in the neocortex provides a substrate for behavioral recovery after cortical damage.
While the GAP-43 and synaptophysin immunoreactivity demonstrate changes in the expression of the protein levels after ischemic damage, there are other explanations for the results seen in this study. It is possible that the immunoreaction product is a result of nonspecific immunoreactivity. However, we minimized any contribution of nonspecific immunostaining in the cortex outside of the infarction by obtaining measurements of immunoreactivity from control tissue and deducting this measurement from primary readings. Another interpretation is that changes in levels of synaptophysin could represent alterations of synaptic terminal size or vesicle number after infarction. For example, larger synaptic terminals or a blocked synaptic release mechanism would result in increased numbers of synaptic vesicles that would be reflected by changes in synaptophysin immunoreactivity. Both of these mechanisms are unlikely since no evidence of these phenomena after CNS trauma exists; however, these alternatives could be tested by the use of electron microscopy and stereological techniques.
Since the area of infarction created by the ischemic insult included the sensorimotor cortex representation of the forelimb, it was important to determine whether any behavioral changes occurred in the contralateral forelimb function as a result of the ischemia and whether the forelimb behavioral dysfunction recovers over time. Two sizes of grids were used for the forelimb foot-fault test, and both sizes indicated statistically significant changes in contralateral forelimb behavior as a result of the neocortical ischemia. The data of the behavioral recovery over time, however, indicate that the large grid was not as sensitive as the small grid in terms of demonstrating functional recovery. Nevertheless, the forelimb recovery on the side contralateral to the neocortical stroke demonstrates a functional recovery in the same animals in which the immunoreaction indicates a neocortical reorganization in the forelimb regions. The contralateral cortex is postulated to play a role in the forelimb recovery since upregulation of synaptophysin is demonstrated contralateral as well as ipsilateral to the neocortical infarct.
It is of interest to compare our results with those obtained after a thrombotic infarction of the vibrissal barrel-field cortex, which resides in the parietal neocortices, to emphasize the importance of the contralateral cortex. In unilaterally infarcted animals, the ability to respond to vibrissal sensory information with a motor task was impaired but recovered over time by 60 days. Bilaterally infarcted animals demonstrated no recovery.44 These data support the involvement of the intact contralateral cortex in the recovery of function. Furthermore, 2-deoxyglucose studies by the same group indicated that 30 days after the unilateral cortical infarction, activation of the vibrissae contralateral to the infarct resulted in a spread of activation anterior and lateral to the infarcted zone and included areas within the ipsilateral somatosensory cortex.45 Our studies confirm the involvement of the neocortex anteriorly, medial and lateral to the infarct and contralateral to the infarct, in that proteins known to be involved in neuroanatomical remodeling are upregulated in these regions. Thus, the present studies confirm earlier studies that behavioral recovery in unilaterally infarcted animals is a consequence of functional plasticity with resultant neural circuit reorganization and extend these findings to propose that neuroanatomical reorganization is one mechanism by which neural circuit reorganization is achieved.46
Increased dendritic branching in the cortex visualized with Golgi staining techniques has been reported after hemidecortications and unilateral motor and bilateral medial frontal cortex lesions in rats with enriched environments and in aging humans and in some cases is accompanied by behavioral recovery.47 48 49 50 51 52 Changes in dendritic branching could help to promote long-term changes in synaptic functional maps in surrounding focal ischemic lesions or alterations of peripheral nerve input such as those that have been reported with the use of electrophysiological tracking in primates.53 54 Work by Jones and Schallert37 38 shows that after electrolytic cortical lesions in rats, the layer V pyramidal cells in the somatosensory cortex of the contralateral hemisphere show increased dendritic branching, which is dependent on increased usage of the forelimb ipsilateral to the lesion and must include the lesion as a stimulus; ie, increased use itself is not enough to "force" dendritic branching. Thus, both denervation and functional activation appear to be requirements for improved functional recovery.
Lesions of the CNS must initiate a signal for upregulation of the necessary proteins if synaptic remodeling is to occur. In the present study GAP-43 is used to indicate the distribution of neurite growth cones, which become axonal growth cones. However, in vitro experiments with cultured hippocampal neurons demonstrate that before the establishment of axonal polarity, GAP-43 is distributed equally among the growth cones of all neurites and then is attenuated in dendrites once axonal polarity is established.55 56 Since GAP-43 is expressed in dendrites in a very early and narrow temporal window (30 minutes) and is later expressed only in axonal growth cones, the present study supports the concept of cortical neurite plasticity in both dendritic and axonal populations in response to cortical infarction. In addition, continued behavioral tests could provide the activation necessary to stimulate neuronal plasticity in alternate neuronal pathways appropriate for functional restoration. Consequently, cortical plasticity through a variety of mechanisms is becoming well documented and provides a basis for functional recovery, presumably by modification in neural circuits.
Neuronal sprouting occurs during the development of the CNS. However, it is necessary for mature neurons to sprout and establish new connections in the CNS during adult life because of changes in circuitry resulting from environmental changes or cell death.1 2 Following large-scale neuronal death, as in ischemia, regions of surviving neurons may reorganize in response to the denervation. This anatomic plasticity would result in alterations of neuronal pathways. While anatomic and electrophysiological reorganization have been well documented following lesions of the immature CNS1 2 51 and pathways of the adult hippocampus,18 34 little research has been conducted in the anatomic plasticity of the mature cortex after damage. It should be noted that electrophysiological evidence for cortical plasticity after digit amputation, peripheral nerve sectioning, and focal ischemia in primates demonstrated changes both acutely and chronically,53 but no neuroanatomical techniques were used to determine whether new, alternate pathways had formed.
While most stroke survivors have some form of permanent disability, they generally show recovery from behavioral dysfunction after stroke.57 Normally, there is spontaneous recovery of some disabilities within days after infarction58 and improvement of residual deficits in the first 3 to 6 months after damage.59 The short-term recovery may be due to the resolution of pathological changes from the stroke such as edema, swelling of the brain, and diaschisis, which is a metabolic and electrophysiological depression of seemingly healthy neurons in regions away from damage.60 The short-term recovery is followed by a more gradual recovery over the following months that tends to plateau at 1 year. Mechanisms of this long-term recovery are not fully understood.
There are several hypotheses for the observed long-term recovery in patients and animal models: (1) the resolution of chronic diaschisis; it is possible that areas of the brain could remain inactive for extended periods of time and that the mechanism of recovery is simply the resolution of chronic diaschisis; (2) the strengthening of secondary pathways in the brain; the CNS has many redundant pathways, and if the primary pathway in the CNS is damaged it is possible that a secondary pathway could carry messages in the brain, allowing for the reestablishment of behavior; (3) the substitution of an alternative behavior acquired over repeated trials; the inclusion of the sham control animals as well as preoperative behavioral testing should control for this possibility; (4) the activation of "silent synapses"; connections between neurons have different "strengths," and some neurons are more effective at transmitting action potentials to targeted dendrites than others. It is possible that if the stronger neurons were removed, the weaker neurons would have a greater control over their targets61 ; and (5) anatomic plasticity; when neurons are removed from the neuropil, vacancies appear on dendritic arbors, and axons from uninjured neurons sprout and form synapses to fill the empty spaces. The data in the present study demonstrate that anatomic plasticity occurs in the neocortex, providing a mechanism for behavioral recovery after stroke.
Past scientific investigations held that the cortex is not capable of plasticity but is hardwired and immutable. Once damage occurred, cortical neurons either died or at best did not change their projection patterns.2 However, a great deal of evidence suggests otherwise. To our knowledge, this report is the first to describe an increase in GAP-43–like immunoreactivity followed by an increase in synaptophysin immunoreactivity in areas of the neocortex, both ipsilaterally and contralaterally, that are in the appropriate neocortical regions and correlate with an improved behavioral function. These findings support the involvement of axonal sprouting followed by synaptogenesis in the neocortex after focal ischemia as a mechanism for the recovery of forelimb function described in the present study as well as in other laboratories.62 63 Furthermore, an understanding of the progression of molecular events that provide the basis for remodeling in the cortex after stroke will allow the development of exogenous therapies to improve neuroanatomical plasticity after infarction, which may promote behavioral recovery.
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Acknowledgments |
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This study was supported by NS-07185, NS-11255, RR-03979, and Bristol-Myers Squibb. The authors would like to thank Drs William D. Willis and Myron D. Ginsberg for their careful reading and constructive criticism of this manuscript.
Received March 30, 1995; revision received August 16, 1995; accepted August 17, 1995.
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 P. Chen, D. E. Goldberg, B. Kolb, M. Lanser, and L. I. Benowitz Inosine induces axonal rewiring and improves behavioral outcome after stroke PNAS, June 25, 2002; 99(13): 9031 - 9036. [Abstract] [Full Text] [PDF]
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H. Bolay, Y. Gursoy-Ozdemir, Y. Sara, R. Onur, A. Can, and T. Dalkara Persistent Defect in Transmitter Release and Synapsin Phosphorylation in Cerebral Cortex After Transient Moderate Ischemic Injury Stroke, May 1, 2002; 33(5): 1369 - 1375. [Abstract] [Full Text] [PDF]
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S. Knecht, T. Imai, S. Kamping, C. Breitenstein, H. Henningsen, B. Lutkenhoner, and E. B. Ringelstein D-amphetamine does not improve outcome of somatosensory training Neurology, December 26, 2001; 57(12): 2248 - 2252. [Abstract] [Full Text] [PDF]
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M. L. Cuadrado, J. A. Arias, W. R. Staines, W. E. McIlroy, S. J. Graham, and S. E. Black Bilateral movement enhances ipsilesional cortical activity in acute stroke: A pilot functional MRI study Neurology, November 13, 2001; 57(9): 1740 - 1741. [Full Text] [PDF]
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S. Watanabe, J. R. Hoffman, R. L. Craik, P. J. Hand, S. E. Croul, M. Reivich, and J. H. Greenberg A New Model of Localized Ischemia in Rat Somatosensory Cortex Produced by Cortical Compression Stroke, November 1, 2001; 32(11): 2615 - 2623. [Abstract] [Full Text] [PDF]
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R. M. Dijkhuizen, J. Ren, J. B. Mandeville, O. Wu, F. M. Ozdag, M. A. Moskowitz, B. R. Rosen, and S. P. Finklestein Functional magnetic resonance imaging of reorganization in rat brain after stroke PNAS, October 12, 2001; (2001) 231235598. [Abstract] [Full Text] [PDF]
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L. Wei, J. P. Erinjeri, C. M. Rovainen, and T. A. Woolsey Collateral Growth and Angiogenesis Around Cortical Stroke Stroke, September 1, 2001; 32(9): 2179 - 2184. [Abstract] [Full Text] [PDF]
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J. Biernaskie and D. Corbett Enriched Rehabilitative Training Promotes Improved Forelimb Motor Function and Enhanced Dendritic Growth after Focal Ischemic Injury J. Neurosci., July 15, 2001; 21(14): 5272 - 5280. [Abstract] [Full Text] [PDF]
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M. J. Hasbani, S. M. Underhill, G. De Erausquin, and M. P. Goldberg Synapse Loss and Regeneration: A Mechanism for Functional Decline and Recovery after Cerebral Ischemia? Neuroscientist, April 1, 2000; 6(2): 110 - 119. [Abstract] [PDF]
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B. B. Johansson Brain Plasticity and Stroke Rehabilitation : The Willis Lecture Stroke, January 1, 2000; 31(1): 223 - 230. [Abstract] [Full Text] [PDF]
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H. Yamauchi, H. Fukuyama, Y. Nagahama, S. Nishizawa, and J. Konishi Uncoupling of Oxygen and Glucose Metabolism in Persistent Crossed Cerebellar Diaschisis Stroke, July 1, 1999; 30(7): 1424 - 1428. [Abstract] [Full Text] [PDF]
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L. B. Goldstein and C. E. Hulsebosch Amphetamine-Facilitated Poststroke Recovery • Response Stroke, March 1, 1999; 30(3): 696 - 698. [Full Text] [PDF]
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R. P. Stroemer, T. A. Kent, C. E. Hulsebosch, and D. M. Feeney Enhanced Neocortical Neural Sprouting, Synaptogenesis, and Behavioral Recovery With D-Amphetamine Therapy After Neocortical Infarction in Rats • Editorial Comment Stroke, November 1, 1998; 29(11): 2381 - 2395. [Abstract] [Full Text] [PDF]
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F. C. Barone, R. F. White, P. A. Spera, J. Ellison, R. W. Currie, X. Wang, G. Z. Feuerstein, and N. J. Rothwell Ischemic Preconditioning and Brain Tolerance: Temporal Histological and Functional Outcomes, Protein Synthesis Requirement, and Interleukin-1 Receptor Antagonist and Early Gene Expression • Editorial Comment Stroke, September 1, 1998; 29(9): 1937 - 1951. [Abstract] [Full Text] [PDF]
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Y. Li, N. Jiang, C. Powers, M. Chopp, and B. B. Johansson Neuronal Damage and Plasticity Identified by Microtubule-Associated Protein 2, Growth-Associated Protein 43, and Cyclin D1 Immunoreactivity After Focal Cerebral Ischemia in Rats • Editorial Comment Stroke, September 1, 1998; 29(9): 1972 - 1981. [Abstract] [Full Text] [PDF]
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H.-J. Bidmon, K. Kato, A. Schleicher, O. W. Witte, K. Zilles, and R. J. Traystman Transient Increase of Manganese–Superoxide Dismutase in Remote Brain Areas After Focal Photothrombotic Cortical Lesion • Editorial Comment Stroke, January 1, 1998; 29(1): 203 - 211. [Abstract] [Full Text] [PDF]
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T. Kawamata, W. D. Dietrich, T. Schallert, J. E. Gotts, R. R. Cocke, L. I. Benowitz, and S. P. Finklestein Intracisternal basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction PNAS, July 22, 1997; 94(15): 8179 - 8184. [Abstract] [Full Text] [PDF]
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D. A. Lin and S. P. Finklestein {blacksquare} REVIEW : Basic Fibroblast Growth Factor: A Treatment for stroke? Neuroscientist, July 1, 1997; 3(4): 247 - 250. [Abstract] [PDF]
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S. C. Cramer, R. L. Robertson, E. C. Dooling, and R. M. Scott Moyamoya and Down Syndrome: Clinical and Radiological Features Stroke, November 1, 1996; 27(11): 2131 - 2135. [Abstract] [Full Text]
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S. Iglesias, G. Marchal, P. Rioux, V. Beaudouin, J.L. Hauttement, V. de la Sayette, F. Le Doze, J.M. Derlon, F. Viader, and J.C. Baron Do Changes in Oxygen Metabolism in the Unaffected Cerebral Hemisphere Underlie Early Neurological Recovery After Stroke?: A Positron Emission Tomography Study Stroke, July 1, 1996; 27(7): 1192 - 1199. [Abstract] [Full Text]
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R. M. Dijkhuizen, J. Ren, J. B. Mandeville, O. Wu, F. M. Ozdag, M. A. Moskowitz, B. R. Rosen, and S. P. Finklestein Functional magnetic resonance imaging of reorganization in rat brain after stroke PNAS, October 23, 2001; 98(22): 12766 - 12771. [Abstract] [Full Text] [PDF]
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