David Dornbos

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 metabolism after forced or voluntary physical exercise.

The pathophysiology of stroke, a leading cause of morbidity and mortality, is still in the process of being understood. Pre-ischemic exercise has been known to be beneficial in reducing the severity of stroke-induced brain injury in animal models. Forced exercise with a stressful component, rather than voluntary exercise, was better able to induce neuroprotection. This study further determined the changes in cerebral metabolism resulting from the two methods of exercise (forced versus voluntary). Adult male Sprague-Dawley rats were randomly assigned to 3 groups: the control group (no exercise), the forced treadmill exercise group, and the voluntary running wheel exercise group. In order to measure the extent of cerebral metabolism in animals with different exercise regimens, mRNA levels and protein expression of glucose transporter 1 and glucose transporter 3 (GLUT-1 and GLUT-3), phosphofructokinase (PFK), lactate dehydrogenase (LDH), and adenosine monophosphate kinase (AMPK) were measured utilizing real-time reverse transcription polymerase chain reaction (PCR) analysis as well as Western blot analysis. Phosphorylated AMPK activity was also measured using an ELISA activity kit, and hypoxic inducible factor (HIF)-1α was measured at transcription and translation levels. The data show that the forced exercise group had a significant (p < 0.05) increase in cerebral glycolysis, including expressions of GLUT-1, GLUT-3, PFK, LDH, phosphorylated AMPK activity and HIF-1α, when compared to the voluntary exercise and the control groups. Our results suggest that the effects of different exercise on HIF-1α expression and cerebral glycolysis may provide a possible reason for the discrepancy in neuroprotection, with forced exercise faring better than voluntary exercise through increased cerebral metabolism.

Remote ischemic post-conditioning reduced brain damage in experimental ischemia/reperfusion injury

Abstract Objectives: To determine the protective effects of remote post-conditioning on ischemic brain lesions caused by middle cerebral artery (MCA) occlusion in rats. Methods: A total of 54 animals were used in this present study. An ischemic stroke model was generated by 90-minute occlusion of right MCA (n = 42). Twelve rats were used as control for studying edema and blood-brain barrier (BBB) integrity. Remote post-conditioning was conducted immediately after MCA occlusion in the bilateral lower limb by occluding and releasing the femoral artery for three cycles; each occlusion and release lasted for 10 minutes. After 24 hours of reperfusion, the cerebral infarct volumes were quantified by 2,3,4-triphenytetrazolium-chloride, brain water content was determined by dry/wet weight method, and damage to the BBB was determined by Evans blue extravasation. Results: Remote post-conditioning significantly reduced brain infarct damage (P<0·0001). Brain edema was significantly (P<0·01) reduced after stroke in the remote post-conditioning group. BBB leakage was significantly reduced in the remote post-conditioning group when compared to the control ischemic groups (P<0·05). Conclusion: These results provide evidence that remote post-conditioning, which was initiated after ischemia and before reperfusion, protects against brain injury in experimental ischemic stroke.

Hypoxia-Inducible Factor-1α Contributes to Brain Edema after Stroke by Regulating Aquaporins and Glycerol Distribution in Brain

Brain edema following stroke is a critical clinical problem due to its association with increased morbidity and mortality. Despite its significance, present treatment for brain edema simply provides symptomatic relief due to the fact that molecular mechanisms underlying brain edema remain poorly understood. The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α) and aquaporins (AQP-4 and -9) in regulating cerebral glycerol accumulation and inducing brain edema in a rodent model of stroke. Two-hours of middle cerebral artery occlusion (MCAO) followed by reperfusion was performed in male Sprague-Dawley rats (250-280 g). Anti-AQP-4 antibody, anti-AQP-9 antibody, or 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α) was given at the time of MCAO. The rats were sacrificed at 1 and 24 hours after reperfusion and their brains were examined. Extracellular and intracellular glycerol concentration of brain tissue was calculated with an enzymatic glycerol assay. The protein expressions of HIF-1α, AQP-4 and AQP-9 were determined by Western blotting. Brain edema was measured by brain water content. Compared to control, edema (p < 0.01), increased glycerol (p < 0.05), and enhanced expressions of HIF-1α, AQP-4, and AQP-9 (p < 0.05) were observed after stroke. With inhibition of AQP-4, AQP-9 or HIF-1α, edema and extracellular glycerol were significantly (p < 0.01) decreased while intracellular glycerol was increased (p < 0.01) 1 hour after stroke. Inhibition of HIF-1α with 2ME2 suppressed (p < 0.01) the expression of AQP-4 and AQP-9. These findings suggest that HIF-1α serves as an upstream regulator of cerebral glycerol concentrations and brain edema via a molecular pathway involving AQP-4 and AQP-9. Pharmacological blockade of this pathway in stroke patients may provide novel therapeutic strategies.

Combined effect of tumor necrosis factor (TNF)-α and heat shock protein (HSP)-70 in reducing apoptotic injury in hypoxia: A cell culture study

Studies have demonstrated neuroprotective effects of either TNF-alpha or HSP-70 in ischemia/reperfusion injury following exercise. However, the protective mechanisms involving combined effect of the two proteins, particularly in neuronal apoptosis, remain unclear. This study aims to elucidate the beneficial role of TNF-alpha and HSP-70 in the regulation of apoptotic proteins and ERK signaling in hypoxic injury. Cortical neurons from 20 Sprague-Dawley rat embryos were isolated and cultured in five groups with or without pretreatment with recombinant TNF-alpha, HSP-70 protein or both prior to hypoxic conditions: (1) control; (2) control/hypoxia; (3) TNF-alpha/hypoxia; (4) HSP-70/hypoxia and (5) TNF-alpha/HSP-70/hypoxia. Western blotting was used to detect pro- and anti-apoptotic proteins, including Bax, AIF, Bcl-xL, Bcl-2, and pERK1/2 protein. TNF-alpha and HSP-70 significantly (p<0.05) reduced the levels of pro-apoptotic proteins, Bax and AIF. Also, pretreatment of hypoxic brain tissue with TNF-alpha and HSP-70 significantly (p<0.05) enhanced the levels of anti-apoptotic protein, Bcl-xL. TNF-alpha and HSP-70 together increased Bcl-2 levels by 70%. Hypoxia caused a significant (p<0.05) increase in ERK1/2 phosphorylation levels by 224%. The most effective inhibition of ERK levels was obtained by the combined administration of TNF-alpha and HSP-70. This study suggested that TNF-alpha and HSP-70 together enhance the decrease in pro-apoptotic protein levels and the increase in anti-apoptotic protein levels in the event of neuronal hypoxia through ERK1/2 signal transduction.

Preischemic exercise reduces brain damage by ameliorating metabolic disorder in ischemia/reperfusion injury

Physical exercise preconditioning is known to ameliorate stroke‐induced injury. In addition to several other mechanisms, the beneficial effect of preischemic exercise following stroke is due to an upregulated capacity to maintain energy supplies. Adult male Sprague‐Dawley rats were used in exercise and control groups. After 1–3 weeks of exercise, several enzymes were analyzed as a gauge of the direct effect of physical exercise on cerebral metabolism. As a measure of metabolic capacity, an ADP/ATP ratio was obtained. Glucose transporters (GLUT1 and GLUT3) were monitored to assess glucose influx, and phosphofructokinase (PFK) was measured to determine the rate of glycolysis. Hypoxia‐induced factor‐1α (HIF‐1α) and 5′AMP‐activated protein kinase (AMPK) levels were also determined. These same analyses were performed on preconditioned and control rats following an ischemic/reperfusion (I/R) insult. Our results show that GLUT1, GLUT3, PFK, AMPK, and HIF‐1α were all increased following 3 weeks of exercise training. In addition, the ADP/ATP ratio was chronically elevated during these 3 weeks. After I/R injury, HIF‐1α and AMPK were significantly higher in exercised rats. The ADP/ATP ratio was reduced in preconditioned rats in the acute phase after stroke, suggesting a lower level of metabolic disorder. GLUT1 and GLUT3 were also increased in the acute phase in exercise rats, indicating that these rats were better able to increase rates of metabolism immediately after ischemic injury. In addition, PFK expression was increased in exercise rats showing an enhanced glycolysis resulting from exercise preconditioning. Altogether, exercise preconditioning increased the rates of glucose metabolism, allowing a more rapid and more substantial increase in ATP production following stroke.

Evaluation of plasma d-dimer plus fibrinogen in predicting acute CVST

Background Prompt diagnosis of cerebral venous sinus thrombosis is a challenge owing in part to its complex and non-specific early clinical symptoms. Objective This study aims to evaluate the value of clinically useful biomarkers (d-dimer and fibrinogen) for cerebral venous sinus thrombosis prediction. Methods Two-hundred and thirty-three suspected cerebral venous sinus thrombosis patients were enrolled in this prospective study. Thirty-four cases confirmed as cerebral venous sinus thrombosis using imaging modalities, whereas the other 199 cases served as mimic controls. Plasma samples of 34 healthy controls were further collected from age- and gender-matched volunteers. d-dimer and fibrinogen levels of all patients and controls were measured before imaging and treatment. The dynamic d-dimer and fibrinogen levels in cerebral venous sinus thrombosis cases after anticoagulation were monitored for up to 180 consecutive days. Results At admission before treatment the average d-dimer and fibrinogen levels in cerebral venous sinus thrombosis group were 968·9 ± 160·1 μg/l and 6·9 ± 1·3 g/l, both of which were significantly elevated when compared with that of the controls. In cerebral venous sinus thrombosis patients, 94·1% had d-dimer elevation, 73·5% had fibrinogen elevation, and 67·6% had both elevated d-dimer and fibrinogen. During acute phase, the sensitivity and specificity of predicting cerebral venous sinus thrombosis using only d-dimer were 94·1% and 97·5%, whereas that of d-dimer in combination with fibrinogen were 67·6% and 98·9%. After administering anticoagulation, d-dimer levels gradually recovered; however, fibrinogen levels fluctuated with 33·3% of the patients still exhibiting elevated values up until 180 days. Conclusion d-dimer may serve as an important screening tool to determine the urgency of obtaining magnetic resonance imaging/magnetic resonance venography or digital subtraction angiography in patients presenting with clinical symptoms that are suspected of cerebral venous sinus thrombosis. Furthermore, d-dimer in combination with fibrinogen may increase the predictive value of acute cerebral venous sinus thrombosis.

Mechanisms of neuronal damage and neuroprotection underlying ischemia/reperfusion injury after physical exercise.

The effects of exercise pre-conditioning on lessening the impact of ischemia/reperfusion injury provide pivotal information and potential targets for future pharmacological intervention. Exercise induces increased expression of neurotrophic factors, the extracellular matrix (ECM) proteins, integrins, angiogenic factors, as well as tumor necrosis factor (TNF-α) and heat shock proteins (Hsp-70). These factors all directly enhance the neurovascular unit and alleviate the harmful effects following ischemia/reperfusion injury. Furthermore, pre-conditioning decreases expression of matrix metalloproteinase (MMP-9) and Toll-like receptor-4, which ameliorates the inflammatory response and apoptosis following ischemic insult. Perhaps most importantly, exercise pre-conditioning shows a propensity to simultaneously favor cell survival mechanisms and inhibit apoptotic pathways via interactions between TNF-α and Hsp-70, which are regulated by extracellular signal-regulated kinases-1 and -2 (ERK1/2). Finally, chronic exercise preconditioning increases cerebral metabolism, effectively enhancing the neuronal response to increase ATP production following periods of hypoxia. The purpose of this review is to demonstrate the various effects of exercise pre-conditioning on the neural response to ischemia/reperfusion injury as a means of demonstrating potential targets for prevention and treatment of acute ischemic events.

Diffusion tensor imaging and diffusion tensor imaging-fibre tractograph depict the mechanisms of Broca-like and Wernicke-like conduction aphasia

Abstract Objectives: Conduction aphasia is usually considered a result of damage of the arcuate fasciculus, which is subjacent to the parietal portion of the supra-marginal gyrus and the upper part of the insula. It is important to stress that many features of conduction aphasia relate to a cortical deficit, more than a pure disconnection mechanism. In this study, we explore the mechanism of Broca-like and Wernicke-like conduction aphasia by using diffusion tensor imaging (DTI) and diffusion tensor imaging-fibre tractograph (DT-FT). Methods: We enrolled five Broca-like conduction aphasia cases, five Wernicke-like aphasia conduction cases and 10 healthy volunteers residing in Beijing and speaking Mandarin. All are right handed. We analyzed the arcuate fasciculus, Broca’s areas and Wernicke’s areas by DTI and measured fractional anisotrogy (FA). The results of left and right hemispheres were compared in both conduction aphasia cases and volunteers. Then the results of the conduction aphasia cases were compared with those of volunteers. The fibre construction of Broca’s and Wernicke’s areas was also compared by DTI-FT. Results: The FA occupied by the identified connective pathways (Broca’s area, Wernicke’s area and the arcuate fasciculus) in the left hemisphere was larger than that in the right hemisphere in the control group (P<0·05). Among Broca-like conduction aphasia cases, the FA of the left Broca’s area was smaller than that of the right mirror side (P<0·05), and the FA of the left anterior segment of the arcuate fasciculus was smaller than that of right mirror side (P<0·05). On the other hand, among Wernicke-like conduction aphasia patients, the FA of the left Wernicke’s area was smaller than that of right mirror side (P<0·05), and the FA of left posterior segment of arcuate fasciculus was smaller than that of right mirror side (P<0·05). Conclusions: Conduction aphasia results from not only arcuate fasciculus destruction, but also from disruption of the associated cortical areas. Along different segments of the arcuate fasciculus, the characteristics of language disorders of conduction aphasia were different. A lesion involving Broca’s area and the anterior segments of the arcuate fasciculus would lead to Broca-like conduction aphasia, whereas a lesion involved Wernicke’s area and posterior segments of the arcuate fasciculus would lead to Wernicke-like conduction aphasia.

Relationship between transcranial Doppler variables in acute stage and outcome of intracerebral hemorrhage

Abstract Objective: To investigate the characteristics of transcranial Doppler variables in the acute stage of intracerebral hemorrhage (ICH) and its relationship with 14-day death and outcomes at 90 day after onset. Methods: Ninety first-time supratentorial nontraumatic ICH patients were prospectively included. Computed tomography and transcranial Doppler examinations were performed on the first, third, seventh, and fourteenth day after onset. Transcranial Doppler variables were obtained from bilateral middle cerebral arteries. The relationship between ICH outcome and the following variables were analyzed: systolic (V s), diastolic (V d), mean (V m) velocities, and pulsatiliy index (PI) from affected and unaffected hemispheres. Results: Fourteen (15·6%) patients died within 14 days after onset of ICH. The stepwise logistic regression analyses proved presence of intraventricular hemorrhage (OR: 11·91; 95%CI: 1·62-87·42) and PI from unaffected hemisphere (OR: 1·64; 95%CI: 1·19-2·25) to be independent predictors of 14-day death. Forty-eight of the 90 ICH patients performed the transcranial Doppler monitoring at all four time points. V m from both hemispheres decreased gradually within 14 days after onset. Among the 48 patients, 22 patients were dependent (modified Rankin Scale3) at 90 days after onset. Compared with patients who were independent at 90 days, V d (F = 4·98, P = 0·03) and V m (F = 7·30, P<0·01) from unaffected hemisphere were significantly lower, while the PI was significantly higher in patients who were dependent (F = 9·84, P<0·01). Conclusions: Presence of intraventricular hemorrhage and PI from unaffected hemisphere proved to be independent predictors of 14-day death. Sustained persistent decreases in V d and V m and increases in PI from the unaffected hemisphere during the acute stage may be related with dependency at 90 days.