Yuchuan Ding

Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

The role of hypoxia-inducible factor-1α, aquaporin-4, and matrix metalloproteinase-9 in blood-brain barrier disruption and brain edema after traumatic brain injury

OBJECT The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α), aquaporin-4 (AQP-4), and matrix metalloproteinase-9 (MMP-9) in blood-brain barrier (BBB) permeability alterations and brain edema formation in a rodent traumatic brain injury (TBI) model. METHODS The brains of adult male Sprague-Dawley rats (400-425 g) were injured using the Marmarou closed-head force impact model. Anti-AQP-4 antibody, minocycline (an inhibitor of MMP-9), or 2-methoxyestradiol (2ME2, an inhibitor of HIF-1α), was administered intravenously 30 minutes after injury. The rats were killed 24 hours after injury and their brains were examined for protein expression, BBB permeability, and brain edema. Expression of HIF-1α, AQP-4, and MMP-9 as well as expression of the vascular basal lamina protein (laminin) and tight junction proteins (zona occludens-1 and occludin) was determined by Western blotting. Blood-brain barrier disruption was assessed by FITC-dextran extravasation, and brain edema was measured by the brain water content. RESULTS Significant (p < 0.05) edema and BBB extravasations were observed following TBI induction. Compared with sham-operated controls, the injured animals were found to have significantly (p < 0.05) enhanced expression of HIF-1α, AQP-4, and MMP-9, in addition to reduced amounts (p < 0.05) of laminin and tight junction proteins. Edema was significantly (p < 0.01) decreased after inhibition of AQP-4, MMP-9, or HIF-1α. While BBB permeability was significantly (p < 0.01) ameliorated after inhibition of either HIF-1α or MMP-9, it was not affected following inhibition of AQP-4. Inhibition of MMP reversed the loss of laminin (p < 0.01). Finally, while inhibition of HIF-1α significantly (p < 0.05) suppressed the expression of AQP-4 and MMP-9, such inhibition significantly (p < 0.05) increased the expression of laminin and tight junction proteins. CONCLUSIONS The data support the notion that HIF-1α plays a role in brain edema formation and BBB disruption via a molecular pathway cascade involving AQP-4 and MMP-9. Pharmacological blockade of this pathway in patients with TBI may provide a novel therapeutic strategy.

Cerebral angiogenesis and expression of angiogenic factors in aging rats after exercise.

The effect that exercise has on angiogenesis in the aging rat is unknown. We initiated this study with the intent to determine if exercise could induce angiogenesis in aging rats, as well as in adult rats reported previously. The markers we used to determine our endpoint were vascular endothelial growth factor (VEGF) and angiopoietin 1 and 2, as well as vascular density. Aged (22 month old) female Fisher 344 rats (n=16) were exercised on a treadmill 30 minutes each day for 3 weeks, or housed as non-exercised controls for the same duration. At the end of the exercise protocol, a significant (p<0.01) increase in the density of microvessels was found within the cerebral vasculature of the rats. Exercise was also associated with a significantly (p<0.01) increased mRNA expression of angiopoietin 1 and 2 in the aged cohort of rats. A mild but significant (p<0.01) increase in the four isoforms of VEGF mRNA (120, 144, 164, 188) were observed, with VEGF120 and VEGF144 being more markedly up-regulated than the other two. VEGF protein expression was also significantly (p<0.01) increased. This study demonstrates that angiogenesis can be induced in aging rats via exercise. The induced angiogenesis was associated with overexpression of angiogenic factors. These results support the hypothesis that an angiogenic response to chronic physical exercise is maintained with aging.

Exercise-Induced Overexpression of Angiogenic Factors and Reduction of Ischemia / Reperfusion Injury in Stroke

The purpose of this study was to determine if exercise could induce expression of vascular endothelial growth factor (VEGF) and angiopoietin 1 and 2, in association with angiogenesis; and if angiogenic changes correlated with reduced brain injury in stroke. Adult male Sprague Dawley rats (3 month old, n=44) were exercised on a treadmill 30 minutes each day for 1, 3 or 6 weeks, or housed as non-exercised controls for 3 weeks. Some 3 week-exercised rats were then housed for an additional 3 weeks. Exercise significantly (p<0.01) increased mRNA (determined by real-time reverse transcriptase-polymerase chain reaction) expression of angiopoietin 1 and 2 as early as 1 week, with further increases occurring at 3 weeks. A mild increase after 1 week and a robust increase after 3 weeks of exercise in four isoforms (120, 144, 164, 188) of VEGF mRNA levels were significantly (p<0.01) observed, with VEGF(144) being more markedly up-regulated. Overexpression of the mRNAs decreased upon withdrawal of exercise. A significant increase (p<0.01) in the density of microvessels (determined by laminin-immunocytochemistry) was found at 3 weeks of exercise and this continued after exercise was withdrawn. In exercising rats subjected to 2-h MCA occlusion followed by 48-h reperfusion, neurological deficits and infarct volume were significantly reduced. Neuroprotection continued after 3 weeks of rest. This study indicates that pre-ischemic exercise reduces brain injury in stroke. The reduced damage is associated with angiogenesis, possibly induced by angiogenic factors following exercise. Physical exercise up-regulates mRNA levels of the angiopoietin family and VEGF.

Increased astrocyte proliferation in rats after running exercise

The aim in this study was to investigate whether physical exercise could induce astroglial proliferation in the frontoparietal cortex and dorsolateral striatum where extensive angiogenesis had been found after exercise in previous studies. Adult male Sprague Dawley rats (n=48) were used in four experimental groups. Animals were exercised 30 min each day on a treadmill on which repetitive locomotor movement was required, for 0 (n=12), 3 (n=12) or 6 (n=12) weeks, as well as 3-week exercise plus 3-week rest (n=12). Brain tissues of the exercised and non-exercised rats were processed for glial fibrillary acidic protein (GFAP) immunocytochemistry (n=6 x 4) and Western blotting (n=6 x 4) to evaluate regional astrocyte proliferation in the frontoparietal cortex and dorsolateral striatum. By using GFAP immunocytochemistry and stereological methods, we compared the density of astrocytes in the animals with or without exercise. In comparison to non-exercised animals, a significant (p<0.01) increase in the number of astrocytes was observed in both cortex and striatum of rats exercised for 3 or 6 weeks. Our data also indicated that astrocytic density continued to increase up to 6 weeks either with an additional 3 weeks of exercise (p<0.01) or 3 weeks of rest (p<0.01). In addition, Western blotting analysis showed an obvious increase in GFAP protein from cortex and striatum of exercised animals. Astrocytosis after exercise, coupled with angiogenesis, is thought to provide strength to the neurovascular unit (a construct consisting of microvascular endothelium, astroglia, neurons and the extracellular matrix). Strengthening of this unit by exercise may protect blood-brain-barrier function following brain injury, such as that occurring after stroke.

Motor balance and coordination training enhances functional outcome in rat with transient middle cerebral artery occlusion

The goal of this study was to determine if relatively complex motor training on Rota-rod involving balance and coordination plays an essential role in improving motor function in ischemic rats, as compared with simple locomotor exercise on treadmill. Adult male Sprague-Dawley rats with (n=40) or without (n=40) ischemia were trained under each of three conditions: (1) motor balance and coordination training on Rota-rod; (2) simple exercise on treadmill; and (3) non-trained controls. Motor function was evaluated by a series of tests (foot fault placing, parallel bar crossing, rope and ladder climbing) before and at 14 or 28 days after training procedures in both ischemic and normal animals. Infarct volume in ischemic animals was determined with Nissl staining. Compared with both treadmill exercised and non-trained animals, Rota-rod-trained animals with or without ischemia significantly (P<0.01) improved motor performance of all tasks except for foot fault placing after 14 days of training, with normal rats having better performance. Animals trained for up to 28 days on the treadmill did not show significantly improved function. With regard to foot fault placing task, performance on foot placing was improved in ischemic rats across the three measurements at 0, 14 and 28 days regardless of training condition, while the normal group reached their best performance at the beginning of measurement. No significant differences in infarct volume were found in rats trained either with Rota-rod (47+/-4%; mean+/-S.E.), treadmill (45+/-5%) or non-exercised control (45+/-3%). In addition, no obvious difference could be detected in the location of the damage which included the dorso-lateral portion of the neostriatum and the frontoparietal cortex, the main regions supplied by the middle cerebral artery. The data suggest that complex motor training rather than simple exercise effectively improves functional outcome.

Prereperfusion Saline Infusion Into Ischemic Territory Reduces Inflammatory Injury After Transient Middle Cerebral Artery Occlusion in Rats

Background and Purpose— In ischemic stroke, the ischemic crisis activates a cascade of events that are potentiated by reperfusion, eventually leading to cell death. The chief aim in this study was to investigate whether our new experimental model for stroke therapy, flushing the ischemic territory with saline before reperfusion, could minimize this damage by (1) reducing the inflammatory reaction and (2) improving regional microcirculation. Methods— Stroke in Sprague-Dawley rats (n=39) was induced by a 2-hour middle cerebral artery occlusion with the use of a novel intraluminal hollow filament. Before 48-hour reperfusion, 20 of the ischemic rats received 7 mL isotonic saline at 23°C or 37°C infused into the ischemic area through the filament. Regional cerebral blood flow in cortex supplied by the right middle cerebral artery was measured by laser-Doppler flowmetry during ischemia and reperfusion. Leukocyte infiltration, microvascular plugging, and infarct volume were compared with the use of hematoxylin and eosin staining. Expression of intercellular adhesion molecule 1 (ICAM-1) was determined by immunocytochemistry. Neurological deficits were evaluated. Results— After the prereperfusion infusion of saline, significantly (P <0.001) improved cerebral blood flow (105±12% of baseline) was obtained up to 48 hours after reperfusion, compared with 45±7% at 24 hours and 25±3% at 48 hours after reperfusion without local saline infusion. Significant (P <0.001) reductions in leukocyte infiltration (61%), vascular plugging (45%), infarct volume (approximately 65%), and neurological deficits were also produced. ICAM-1 expression in the infarct region was significantly (P <0.05) minimized by 37%. Conclusions— The reduced brain infarct and neurological deficits may be attributed to adequate reperfusion and ameliorated inflammation induced by local prereperfusion infusion.

Long-term neuroprotective effect of inhibiting poly(ADP-ribose) polymerase in rats with middle cerebral artery occlusion using a behavioral assessment

Poly(ADP-ribose) polymerase (PARP) can initiate an energy-consuming and inefficient repair cycle following cerebral ischemia/reperfusion by transferring ADP ribose units to nuclear proteins eventually leading to cellular dysfunction and neuronal death. 3-Aminobenzamide (3-AB) is a selective inhibitor of PARP that can significantly reduce brain damage after focal ischemia in rats and displays a low toxicity in vivo. The goals of this study were to determine if inhibiting PARP with 3-AB has a long-term neuroprotective effect and if functional outcome improves in rats following focal ischemia and treatment with 3-AB. Focal ischemia was induced by a 2-h occlusion of the middle cerebral artery (MCA), using an intraluminal filament. Motor functions were evaluated from 5 to 28 days after reperfusion in four groups of rats: stroke without treatment; stroke treated with 3-AB at doses of 15 mg/kg, stroke treated with 3-AB at doses of 55 mg/kg; and the non-ischemic control rats. Functional behaviors were tested by a series of motor function tasks (foot placing, parallel bar crossing, rope and ladder climbing), as well as a neurological examination. Infarct volume of stroke brain in the same rat was determined by Nissl staining 28 days after surgery. Comparison of the untreated stroke group (n=11) and the treated stroke groups indicates that impairment of motor function was significantly (P<0.001) reduced by administration of 3-AB at doses of 15 mg/kg (n=9) or 55 mg/kg (n=10). Neurological outcome was also improved significantly (P<0.001). Infarct volume was significantly (P<0.01) reduced in both treated groups. Long-term neuroprotection following ischemia/reperfusion injury to the brain can be obtained by administration of a PARP inhibitor. The motor tests employed in this study can be used as sensitive, objective and reproducible measurements of functional impairment in rats following an ischemic stroke.

Local saline infusion into ischemic territory induces regional brain cooling and neuroprotection in rats with transient middle cerebral artery occlusion.

OBJECTIVEThe neuroprotective effect of hypothermia has long been recognized. Use of hypothermia for stroke therapy, which is currently being induced by whole-body surface cooling, has been limited primarily because of management problems and severe side effects (e.g., pneumonia). The goal of this study was to determine whether local infusion of saline into ischemic territory could induce regional brain cooling and neuroprotection. METHODSA novel procedure was used to block the middle cerebral artery of rats for 3 hours with a hollow filament and locally infuse the middle cerebral artery-supplied territory with 6 ml cold saline (20°C) for 10 minutes before reperfusion. RESULTSThe cold saline infusion rapidly and significantly reduced temperature in cerebral cortex from 37.2 ± 0.1 to 33.4 ± 0.4°C and in striatum from 37.5 ± 0.2 to 33.9 ± 0.4°C. The significant hypothermia remained for up to 60 minutes after reperfusion. Significant (P < 0.01) reductions in infarct volume (approximately 90%) were evident after 48 hours of reperfusion. In ischemic rats that received the same amount of cold saline systemically through a femoral artery, a mild hypothermia was induced only in the cerebral cortex (35.3 ± 0.2°C) and returned to normal within 5 minutes. No significant reductions in infarct volume were observed in this group or in the ischemic group with local warm saline infusion or without infusion. Furthermore, brain-cooling infusion significantly (P < 0.01) improved motor behavior in ischemic rats after 14 days of reperfusion. This improvement continued for up to 28 days after reperfusion. CONCLUSIONLocal prereperfusion infusion effectively induced hypothermia and ameliorated brain injury from stroke. Clinically, this procedure could be used in acute stroke treatment, possibly in combination with intra-arterial thrombolysis or mechanical disruption of clot by means of a microcatheter.

Impaired motor learning and diffuse axonal damage in motor and visual systems of the rat following traumatic brain injury

Abstract Cognitive-motor functioning or motor skill learning is impaired in humans following traumatic brain injury. A more complete understanding of the mechanisms involved in disorders of motor skill learning is essential for any effective rehabilitation. The specific goals of this study were to examine motor learning disorders, and their relationship to pathological changes in adult rats with mild to moderate closed head injury. Motor learning deficits were determined by comparing the ability to complete a series of complex motor learning tasks with simple motor activity. The extent of neuronal damage was determined using silver impregnation. At all post-injury time points (day 1 to day 14), statistically significant deficits were observed in parallel bar traversing, foot placing, ladder climbing, and rope climbing. Performance improved with time, but never reached control levels. In contrast, no deficits were found in simple motor activity skills tested with beam balance and runway traverse. Histologically, axonal degeneration was widely distributed in several brain areas that relate to motor learning, including the white matter of sensorimotor cortex, corpus callosum, striatum, thalamus and cerebellum. Additionally, severely damaged axons were observed in the primary visual pathway, including the optic chiasm, optic tract, lateral geniculate nuclei, and superior colliculus. These findings suggest that motor learning deficits could be detected in mild or moderate brain injury, and this deficit could be attributed to a diffuse axonal injury distributed both in the motor and the visual systems. [Neurol Res 2001; 23: 193-202]