Functional magnetic resonance imaging of reorganization in rat brain after stroke.

Functional recovery after stroke has been associated with brain plasticity; however, the exact relationship is unknown. We performed behavioral tests, functional MRI, and histology in a rat stroke model to assess the correlation between temporal changes in sensorimotor function, brain activation patterns, cerebral ischemic damage, and cerebrovascular reactivity. Unilateral stroke induced a large ipsilateral infarct and acute dysfunction of the contralateral forelimb, which significantly recovered at later stages. Forelimb impairment was accompanied by loss of stimulus-induced activation in the ipsilesional sensorimotor cortex; however, local tissue and perfusion were only moderately affected and cerebrovascular reactivity was preserved in this area. At 3 days after stroke, extensive activation-induced responses were detected in the contralesional hemisphere. After 14 days, we found reduced involvement of the contralesional hemisphere, and significant responses in the infarction periphery. Our data suggest that limb dysfunction is related to loss of brain activation in the ipsilesional sensorimotor cortex and that restoration of function is associated with biphasic recruitment of peri- and contralesional functional fields in the brain.

A functional MRI study of three motor tasks in the evaluation of stroke recovery.

Functional brain imaging studies have provided insights into the processes related to motor recovery after stroke. The comparative value of different motor activation tasks for probing these processes has received limited study. We hypothesized that different hand motor tasks would activate the brain differently in controls, and that this would affect control-patient comparisons. Functional magnetic resonance imaging (MRI) was used to evaluate nine control subjects and seven patients with good recovery after a left hemisphere hemiparetic stroke. The volume of activated brain in bilateral sensorimotor cortex and four other motor regions was compared during each of three tasks performed by the right hand: index-finger tapping, four-finger tapping, and squeezing. In control subjects, activation in left sensorimotor cortex was found to be significantly larger during squeezing as compared with index-finger tapping. When comparing control subjects with stroke patients, patients showed a larger volume of activation in right sensorimotor cortex during index-finger tapping but not with four-finger tapping or squeezing. In addition, patients also showed a trend toward larger activation volume than controls within left supplementary motor area during index-finger tapping but not during the other tasks. Motion artifact was more common with squeezing than with the tapping tasks. The choice of hand motor tasks used during brain mapping can influence findings in control subjects as well as the differences identified between controls and stroke patients. The results may be useful for future studies of motor recovery after stroke.

Intravenous basic fibroblast growth factor produces a persistent reduction in infarct volume following permanent focal ischemia in rats.

Basic fibroblast growth factor (bFGF) is a polypeptide with potent survival-promoting and protective effects on brain cells. In previous studies, we showed that intravenous administration of bFGF reduced infarct volume in models of focal cerebral ischemia in rats, mice, and cats. In these previous studies, infarct volume was measured within 1-7days of the onset of ischemia. The current study was undertaken to determine whether the reduction in infarct volume by bFGF was persistent beyond the first week after stroke. Mature male Sprague-Dawley rats received an intravenous infusion of bFGF (50 microg/kg per h) or vehicle during 0.5-3.5h after permanent proximal middle cerebral artery occlusion. We found a 27% reduction in infarct volume in bFGF- compared to vehicle-treated animals at three months after infarction (P<0.05). The data show that intravenous bFGF treatment produces a persistent reduction in infarct volume, at least up to three months following focal stroke.

Intravenous basic fibroblast growth factor (bFGF) decreases DNA fragmentation and prevents downregulation of Bcl-2 expression in the ischemic brain following middle cerebral artery occlusion in rats.

In previous studies, we showed that basic fibroblast growth factor (bFGF) reduced infarct volume when infused intravenously in animal models of focal cerebral ischemia. In the current study, we examined the potential mechanism of infarct reduction by bFGF, especially effects on apoptosis within the ischemic brain. We found that bFGF decreased DNA fragmentation in the ischemic hemisphere, as assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) histochemical methods combined with morphological criteria. bFGF also prevented reduction of immunoreactivity of the anti-apoptotic protein Bcl-2 in the ischemic hemisphere, but did not alter immunoreactivity of the pro-apoptotic proteins Bax, Caspase-1, or Caspase-3. These changes in TUNEL histochemistry and Bcl-2 immunoreactivity were especially prominent in cortex at the borders ('penumbra') of infarcts, spared by bFGF treatment. We conclude that the infarct-reducing effects of bFGF may be due, in part, to prevention of downregulation of Bcl-2 expression and decreased apoptosis in the ischemic brain.

Time window of intracisternal osteogenic protein-1 in enhancing functional recovery after stroke.

Osteogenic protein-1 (OP-1, BMP-7) is a member of the bone morphogenetic protein subfamily of the TGF-ss superfamily that selectively stimulates dendritic neuronal outgrowth. In previous studies, we found that the intracisternal injection of OP-1, starting at one day after stroke, enhanced sensorimotor recovery of the contralateral limbs following unilateral cerebral infarction in rats. In the current study, we further explored the time window during which intracisternal OP-1 enhances sensorimotor recovery, as assessed by limb placing tests. We found that intracisternal OP-1 (10 microg) given 1 and 3 days, or 3 and 5 days, but not 7 and 9 days after stroke, significantly enhanced recovery of forelimb and hindlimb placing. There was no difference in infarct volume between vehicle- and OP-1-treated animals. The mechanism of OP-1 action might be stimulation of new dendritic sprouting in the remaining uninjured brain.

A pilot study of somatotopic mapping after cortical infarct.

BACKGROUND AND PURPOSE: Animal studies have described remodeling of sensory and motor representational maps after cortical infarct. These changes may contribute to return of function after stroke. METHODS: Functional MRI was used to compare sensory and motor maps obtained in 35 normal control subjects with results from 2 patients with good recovery 6 months after a cortical stroke. RESULTS: During finger tapping in controls, precentral gyrus activation exceeded or matched postcentral gyrus activation in 40 of 42 cases. Patient 1 had a small infarct limited to precentral gyrus. Finger tapping activated only postcentral gyrus, a pattern not seen in any control subject. During tactile stimulation of a finger or hand in controls, postcentral gyrus activation exceeded or matched precentral gyrus activation in 11 of 14 cases. Patient 2 had a small infarct limited to postcentral gyrus and superior parietal lobule. Tactile stimulation of the finger activated only precentral gyrus, a pattern not seen in any control. In both patients, activation during pectoralis contraction was medial to the site activated during finger tapping. CONCLUSIONS: Results during finger tapping (patient 1) and finger stimulation (patient 2) may reflect amplification of a preserved component of normal sensorimotor function, a shift in the cortical site of finger representation, or both. Cortical map reorganization along the infarct rim may be an important contributor to recovery of motor and sensory function after stroke. Functional MRI is useful for assessing motor and sensory representational maps.

Intracisternal antisense oligonucleotide to growth associated protein-43 blocks the recovery-promoting effects of basic fibroblast growth factor after focal stroke.

Focal infarction (stroke) of the lateral cerebral cortex of rats (including the sensorimotor cortex) produces deficits in sensorimotor function of the contralateral limbs that recover partially over time. In previous studies, we found that the intracisternal injection of basic fibroblast growth factor (bFGF), a potent neurotrophic growth factor, starting at 1 day after stroke, significantly enhanced recovery of sensorimotor function of the contralateral forelimb and hindlimb. Moreover, immunoreactivity (IR) for growth-associated protein-43 (GAP-43), a molecular marker of new axonal growth, was increased in the intact contralateral sensorimotor cortex following bFGF treatment. In the current study, we found that the intracisternal administration of antisense, but not missense, oligonucleotide to GAP-43 blocked the recovery-enhancing effects of bFGF and blocked the increase in GAP-43 IR in the contralateral cortex. These results suggest that upregulation of GAP-43 expression and consequent enhanced axonal sprouting in intact uninjured parts of the brain are likely mechanisms for the recovery-promoting effects of bFGF.

Basic fibroblast growth factor enhances axonal sprouting after cortical injury in rats.

The trophic factors responsible for initiating and guiding the outgrowth of axons have proven to be elusive throughout most of this century. Entorhinal cortex injury, which denervates the hippocampal formation of rats, induces axonal sprouting by several surviving hippocampal afferents and results in a significant elevation of growth factors, one of which is basic fibroblast growth factor (bFGF). The possibility that bFGF may regulate lesion-induced hippocampal sprouting was examined by making i.v. bFGF infusions into rats with unilateral entorhinal lesions. Basic FGF treatment significantly increased sprouting by the cholinergic septodentate pathway. Thus, the increase in bFGF following central nervous system injury may signal its role in the regulation of injury-related axonal remodeling of a cholinergic pathway.

Potential usefulness of basic fibroblast growth factor as a treatment for stroke.

Within the past few years, a growing body of evidence has accumulated indicating that exogenously administered neurotrophic growth factors may limit the extent of acute ischemic neural injury and enhance functional neurorecovery following stroke. One of the most widely studied growth factor in this regard is basic fibroblast growth factor (bFGF). In preclinical studies, bFGF administered intravenously within hours after the onset of ischemia reduces infarct size, presumably due to direct protection of cells at the borders (penumbra) of cerebral infarction. On the other hand, if bFGF is administered intracisternally starting at one day after ischemia, infarct size is not reduced, but recovery of sensorimotor function of the impaired limbs is increased, presumably due to enhancement of new neuronal sprouting and synapse formation in the intact uninjured brain. Clinical trials of the intravenous administration of bFGF as a cytoprotective agent in acute stroke are in progress. Trials of the delayed administration of bFGF as a recovery-promoting agent in subacute stroke are anticipated.

Activation of distinct motor cortex regions during ipsilateral and contralateral finger movements.

Previous studies have shown that unilateral finger movements are normally accompanied by a small activation in ipsilateral motor cortex. The magnitude of this activation has been shown to be altered in a number of conditions, particularly in association with stroke recovery. The site of this activation, however, has received limited attention. To address this question, functional magnetic resonance imaging (MRI) was used to study precentral gyrus activation in six control and three stroke patients during right index finger tapping, then during left index finger tapping. In each hemisphere, the most significantly activated site (P < 0.001 required) was identified during ipsilateral and during contralateral finger tapping. In the motor cortex of each hemisphere, the site activated during use of the ipsilateral hand differed from that found during use of the contralateral hand. Among the 11 control hemispheres showing significant activation during both motor tasks, the site for ipsilateral hand representation (relative to contralateral hand site in the same hemisphere) was significantly shifted ventrally in all 11 hemispheres (mean, 11 mm), laterally in 10/11 hemispheres (mean, 12 mm), and anteriorly in 8/11 hemispheres (mean, 10 mm). In 6 of 11 hemispheres, tapping of the contralateral finger simultaneously activated both the ipsilateral and the contralateral finger sites, suggesting bilateral motor control by the ipsilateral finger site. The sites activated during ipsilateral and contralateral hand movement showed similar differences in the unaffected hemisphere of stroke patients. The region of motor cortex activated during ipsilateral hand movements is spatially distinct from that identified during contralateral hand movements.

Estradiol protects against ischemic injury.

Clinical studies demonstrate that estrogen replacement therapy in postmenopausal women may enhance cognitive function and reduce neurodegeneration associated with Alzheimer's disease and stroke. This study assesses whether physiologic levels of estradiol prevent brain injury in an in vivo model of permanent focal ischemia. Sprague-Dawley rats were ovariectomized; they then were implanted, immediately or at the onset of ischemia, with capsules that produced physiologically low or physiologically high 17beta-estradiol levels in serum (10 or 60 pg/mL, respectively). One week after ovariectomy, ischemia was induced. Estradiol pretreatment significantly reduced overall infarct volume compared with oil-pretreated controls (mean+/-SD: oil = 241+/-88; low = 139+/-91; high = 132+/-88 mm3); this protective effect was regionally specific to the cortex, since no protection was observed in the striatum. Baseline and ischemic regional CBF did not differ between oil and estradiol pretreated rats, as measured by laser Doppler flowmetry. Acute estradiol treatment did not protect against ischemic injury. Our finding that estradiol pretreatment reduces injury demonstrates that physiologic levels of estradiol can protect against neurodegeneration.

Quantitative assessment of mirror movements after stroke.

BACKGROUND AND PURPOSE: Mirror movements (MM) are involuntary synchronous movements of one limb during voluntary unilateral movements of the opposite limb. We measured MM in stroke and control subjects and evaluated whether MM after stroke are related to motor function. METHODS: Twenty-three patients and 16 control subjects were studied. A computerized dynamometer was used during two squeezing tasks to measure intended movements from the active hand as well as MM from the opposite hand. Motor deficits were measured with the arm motor component of the Fugl-Meyer scale. RESULTS: During paretic hand squeezing, MM in the unaffected hand were detected in 70% (repetitive squeeze) to 78% (sustained squeeze) of stroke patients. For both tasks, this was significantly (P < 0.05) greater than the incidence of MM in the paretic hand or in either hand of control subjects (17% to 44%), except when compared with the incidence of MM in the dominant hand of control subjects (56%; P = 0.17). The incidence of MM in the paretic hand was not significantly different from that seen in either hand of control subjects. Patients with MM in the unaffected hand had significantly greater motor deficit than patients without MM. Patients with MM in the paretic hand had significantly better motor function than patients without MM. CONCLUSIONS: Simultaneously recording motor performances of both hands provides precise information to characterize MM. MM in the unaffected hand and in the paretic hand are associated with different degrees of motor deficit after stroke. Evaluation of MM may be useful for studying mechanisms of stroke recovery.

Intracisternal osteogenic protein-1 enhances functional recovery following focal stroke.

Osteogenic protein-1 (OP-1, BMP-7) is a member of the transforming growth factor-beta (TGF-beta) superfamily that selectively induces dendritic outgrowth from cultured neurons. We injected human recombinant OP-1 (1 or 10 micrograms) or vehicle into the cisterna magna of mature male Sprague-Dawley rats 1 and 4 days after focal cerebral infarction induced by middle cerebral artery (MCA) occlusion. OP-1 treatment was associated with a marked enhancement of recovery of sensorimotor function of the impaired forelimb and hindlimb (contralateral to infarcts) as assessed by limb placing tests. This effect appeared to be dose dependent. There was no difference in infarct volume between OP-1 and vehicle-treated rats. The mechanisms of enhanced recovery by intracisternal OP-1 may include promotion of dendritic sprouting in the intact uninjured brain.

A functional MRI study of subjects recovered from hemiparetic stroke.

BACKGROUND AND PURPOSE: Stroke recovery mechanisms remain incompletely understood, particularly for subjects with cortical stroke, in whom limited data are available. We used functional magnetic resonance imaging to compare brain activations in normal controls and subjects who recovered from hemiparetic stroke. METHODS: Functional magnetic resonance imaging was performed in ten stroke subjects with good recovery, five with deep, and five with cortical infarcts. Brain activation was achieved by index finger-tapping. Statistical parametric activation maps were obtained using a t test and a threshold of P < .001. In five bilateral motor regions, the volume of activated brain for each stroke subject was compared with the distribution of activation volumes among nine controls. RESULTS: Control subjects activated several motor regions. During recovered hand finger-tapping, stroke subjects activated the same regions as controls, often in a larger brain volume. In the unaffected hemisphere, sensorimotor cortex activation was increased in six of nine stroke subjects compared with controls. Cerebellar hemisphere contralateral and premotor cortex ipsilateral to this region, as well as supplementary motor areas, also had increased activation. In the stroke hemisphere, activation exceeding controls was uncommon, except that three of five cortical strokes showed peri-infarct activation foci. During unaffected hand finger-tapping, increased activation by stroke subjects compared with controls was uncommon; however, decreased activation was seen in unaffected sensorimotor cortex, suggesting that this region's responsiveness increased to the ipsilateral hand and decreased to contralateral hand movements. Use of a different threshold for defining activation (P < .01) did not change the overall findings (kappa = .75). CONCLUSIONS: Recovered finger-tapping by stroke subjects activated the same motor regions as controls but to a larger extent, particularly in the unaffected hemisphere. Increased reliance on these motor areas may represent an important component of motor recovery. Functional magnetic resonance imaging studies of subjects who recovered from stroke provide evidence for several processes that may be related to restoration of neurologic function.

Computerized measurement of motor performance after stroke.

BACKGROUND AND PURPOSE: Stroke scales usually convert motor status to a score along an ordinal scale and do not provide a permanent recording of motor performance. Computerized methods sensitive to small changes in neurological status may be of value for studying and measuring stroke recovery. METHODS: We developed a computerized dynamometer and tested 23 stroke subjects and 12 elderly control subjects on three motor tasks: sustained squeezing, repetitive squeezing, and index finger tapping. For each subject, scores on the Fugl-Meyer and National Institutes of Health stroke scales were also obtained. RESULTS: Sustained squeezing by the paretic hand of stroke subjects was weaker (9.2 kg) than the unaffected hand (20.2 kg; P < .0005), as well as control dominant (23.1 kg; P < .0005) and nondominant (19.9 kg; P < .005) hands. Paretic index finger tapping was slower (2.5 Hz) than the unaffected hand (4.2 Hz; P < .01), as well as control dominant (4.7 Hz; P < .0005) and nondominant (4.9 Hz; P < .0005) hands. Many features of dynamometer data correlated significantly with stroke subjects' Fugl-Meyer scores, including sustained squeeze maximum force (rho = .91) and integral of force over 5 seconds (rho = .91); repetitive squeeze mean force (rho = .92) and mean frequency (rho = .73); and index finger tap mean frequency (rho = .83). Correlation of these motor parameters with National Institutes of Health stroke scale score was weaker in all cases, a consequence of the scoring of nonmotor deficits on this scale. Dynamometer measurements showed excellent interrater (r = .99) and intrarater (r = .97) reliability. CONCLUSIONS: The degree of motor deficit quantitated with the dynamometer is strongly associated with the extent of neurological abnormality measured with the use of two standardized stroke scales. The computerized dynamometer rapidly measures motor function along a continuous, linear scale and produces a permanent recording of hand motor performance accessible for subsequent analyses.

Postischemic infusion of Cu/Zn superoxide dismutase or SOD:Tet451 reduces cerebral infarction following focal ischemia/reperfusion in rats.

Oxygen-free radicals play a major role in neuronal cell injury following cerebral ischemia/reperfusion. The free-radical scavenging enzyme, Cu/Zn superoxide dismutase (SOD-1), ameliorates various types of brain injury resulting from temporary CNS ischemia. We have compared the cerebroprotective properties of human SOD-1 (hSOD-1) with a novel recombinant SOD-1 hybrid protein, SOD:Tet451, composed of hSOD-1 linked to the neuronal binding fragment of tetanus toxin (TTxC). Following 2 h of temporary middle cerebral artery occlusion, rats infused with equivalent activities of either hSOD-1 or SOD:Tet451 for the initial 3 h of reperfusion showed reductions in cerebral infarct volume of 43 and 57%, respectively, compared to saline-treated controls (P < 0.01). Serum hSOD-1 concentrations in rats receiving SOD:Tet451 were seven-fold higher than those in rats receiving the native enzyme. Animals treated with SOD:Tet451 also demonstrated an extended persistence of hSOD-1 in the bloodstream during drug washout as compared to animals given free enzyme. Immunohistochemical examination of brain sections from an SOD:Tet451-treated ischemic rat showed positive immunoreactivity in the ipsilateral cerebral cortex using either anti-TTxC or anti-human SOD-1 antibodies. Our results document that both hSOD-1 and SOD:Tet451 significantly reduce brain infarct volume in a model of transient focal ischemia/reperfusion in rats. Additionally, our findings suggest that the cerebroprotective effects of SOD-1 may be enhanced by neuronal targeting as seen with the hybrid protein SOD:Tet451.

Intracisternal basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction.

Focal cerebral infarction (stroke) due to unilateral occlusion of the middle cerebral artery in mature rats produces deficits in sensorimotor function of the contralateral limbs that recover partially over time. We found that biweekly intracisternal injection of basic fibroblast growth factor (bFGF; 0.5 microg/injection), a potent neurotrophic polypeptide, markedly enhanced recovery of sensorimotor function of the contralateral limbs during the first month after stroke without apparent adverse side effects. Immunostaining for growth-associated protein 43 (GAP-43), a molecular marker of axonal sprouting, showed a selective increase in GAP-43 immunoreactivity in the intact sensorimotor cortex contralateral to cerebral infarcts following bFGF treatment. These results show that bFGF treatment can enhance functional recovery after stroke, and that the mechanism may include stimulation of neuronal sprouting in the intact brain.

Time window of infarct reduction by intravenous basic fibroblast growth factor in focal cerebral ischemia.

Basic fibroblast growth factor (bFGF) is a heparin-binding polypeptide with potent trophic and protective effects on brain neurons, glia and endothelia. In previous studies, we showed that intravenously administered bFGF reduced the volume of cerebral infarcts following permanent occlusion of the middle cerebral artery in rats. In the current study, we examined the time dependence of bFGF infusion on infarct reduction, and the effect of co-infusion of bFGF with heparin. We found a significant reduction in infarct volume when the bFGF infusion (50 microg/kg per h for 3 h) was begun up to 3 h, but not 4 h after the onset of ischemia. The infarct reducing effects of bFGF were not altered by co-infusion of heparin. These results are potentially important in light of the ongoing clinical trials of intravenous bFGF in acute stroke.

Delayed administration of basic fibroblast growth factor (bFGF) attenuates cognitive dysfunction following parasagittal fluid percussion brain injury in the rat.

The present study evaluates the therapeutic effects of delayed administration of bFGF on cognitive dysfunction and histopathological damage following lateral fluid-percussion (FP) brain injury. Male Sprague-Dawley rats were trained to learn a visuospatial task in a Morris Water Maze (MWM) paradigm and then were anesthetized and subjected to either FP brain injury of moderate severity (2.5-2.8 atm, n = 32) or surgery without brain injury (n = 10). Twenty-four hours postinjury, an infusion cannula connected to a mini-osmotic pump was implanted into the area of maximal cortical injury to continuously infuse either bFGF (2.0 g) or vehicle for 7 days. Treatment with bFGF significantly attenuated posttraumatic memory dysfunction in the MWM at 8 days postinjury when compared to vehicle treatment (p < 0.05). The cortical lesion and significant cell loss in the ipsilateral CA3 region of the hippocampus, produced by FP injury, was not affected by bFGF treatment. However, immunohistochemical evaluation of glial fibrillary acidic protein revealed a trend toward increased astrocytosis in the injured cortex of bFGF-treated animals compared to vehicle-treated animals (p < 0.1). These results indicate that bFGF may be efficacious in attenuating cognitive dysfunction associated with traumatic brain injury.