Middle cerebral artery occlusion (MCAO) in rats is a well-studied experimental model for ischemic stroke leading to brain infarction and functional deficits. Many preclinical studies have focused on a small time window after the ischemic episode to evaluate functional outcome for screening therapeutic candidates. Short evaluation periods following injury have led to significant setbacks due to lack of information on the delayed effects of treatments, as well as short-lived and reversible neuroprotection, so called false-positive results. In this report, we evaluated long-term functional deficit for 90 days after MCAO in two rat strains with two durations of ischemic insult, in order to identify the best experimental paradigm to assess injury and subsequent recovery. Behavioral outcomes were measured pre-MCAO followed by weekly assessment post-stroke. Behavioral tests included the 18-point composite neurological score, 28-point neuroscore, rearing test, vibrissae-evoked forelimb placing test, foot fault test and the CatWalk. Brain lesions were assessed to correlate injury to behavior outcomes at the end of study. Our results indicate that infarction volume in Sprague-Dawley rats was dependent on occlusion duration. In contrast, the infarction volume in Wistar rats did not correlate with the duration of ischemic episode. Functional outcomes were not dependent on occlusion time in either strain; however, measureable deficits were detectable long-term in limb asymmetry, 18- and 28-point neuroscores, forelimb placing, paw swing speed, and gait coordination. In conclusion, these behavioral assays, in combination with an extended long-term assessment period, can be used for evaluating therapeutic candidates in preclinical models of ischemic stroke.
Ischemic stroke; CatWalk; long-term functional recovery; middle cerebral artery occlusion; rat strain
Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after sub-acute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human VEGF (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair, and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier (BBB) integrity and suppression of inflammation was followed by a delayed spatio-temporal regulated increase in neovascularization. These events coincided with a bi-modal pattern of functional recovery: an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multi-modal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.
angiogenesis; blood brain barrier; dystroglycan; inflammation; Avastin
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. Gene expression profiling of blood has led to the identification of stroke biomarkers, and may help identify BAVM biomarkers and illuminate BAVM pathogenesis. It is unknown whether blood gene expression profiles differ between 1) BAVM patients and healthy controls, or 2) unruptured and ruptured BAVM patients at presentation. We characterized blood transcriptional profiles in 60 subjects (20 unruptured BAVM, 20 ruptured BAVM, and 20 healthy controls) using Affymetrix whole genome expression arrays. Expression differences between groups were tested by ANOVA, adjusting for potential confounders. Genes with absolute fold change ≥ 1.2 (false discovery rate corrected p ≤ 0.1) were selected as differentially expressed and evaluated for over-representation in KEGG biological pathways (p ≤ 0.05). Twenty-nine genes were differentially expressed between unruptured BAVM patients and controls, including 13 which may be predictive of BAVM. Patients with ruptured BAVM compared to unruptured BAVM differed in expression of 1490 genes, with over-representation of genes in 8 pathways including MAPK, VEGF, Wnt signaling and several inflammatory pathways. These results suggest clues to the pathogenesis of BAVM and/or BAVM rupture and point to potential biomarkers or new treatment targets.
arteriovenous malformation; blood; gene expression; intracranial hemorrhage; microarray analysis
Background and Purpose
Determining the presence and adequacy of collateral blood flow is important in cerebrovascular disease. Therefore, we explored whether a noninvasive imaging modality, arterial spin labeling (ASL) MRI, could be used to detect the presence and intensity of collateral flow using digital subtraction angiography (DSA) and stable xenon CT cerebral blood flow as gold standards for collaterals and cerebral blood flow, respectively.
ASL and DSA were obtained within 4 days of each other in 18 patients with Moyamoya disease. Two neurointerventionalists scored DSA images using a collateral grading scale in regions of interest corresponding to ASPECTS methodology. Two neuroradiologists similarly scored ASL images based on the presence of arterial transit artifact. Agreement of ASL and DSA consensus scores was determined, including kappa statistics. In 15 patients, additional quantitative xenon CT cerebral blood flow measurements were performed and compared with collateral grades.
The agreement between ASL and DSA consensus readings was moderate to strong, with a weighted kappa value of 0.58 (95% confidence interval, 0.52–0.64), but there was better agreement between readers for ASL compared with DSA. Sensitivity and specificity for identifying collaterals with ASL were 0.83 (95% confidence interval, 0.77–0.88) and 0.82 (95% confidence interval, 0.76–0.87), respectively. Xenon CT cerebral blood flow increased with increasing DSA and ASL collateral grade (P<0.05).
ASL can noninvasively predict the presence and intensity of collateral flow in patients with Moyamoya disease using DSA as a gold standard. Further study of other cerebrovascular diseases, including acute ischemic stroke, is warranted.
angiography; arterial spin labeling; cerebral blood flow; cerebral hemodynamics; cerebrovascular disease; collateral flow; neuroradiology; perfusion
Retrospective studies suggest that p53 alteration is prognostic for recurrence in patients with urothelial bladder cancer and predictive for benefit from combination methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) adjuvant chemotherapy.
Patients and Methods
Patients with pT1/T2N0M0 disease whose tumors demonstrated ≥ 10% nuclear reactivity on centrally performed immunohistochemistry for p53 were offered random assignment to three cycles of adjuvant MVAC versus observation; p53-negative patients were observed. By using a log-rank test with one-sided α = .05 and β = .10, 190 p53-positive patients were planned to be randomly assigned to detect an absolute improvement in probability of recurring by 3 years from 0.50 to 0.30.
A total of 521 patients were registered, 499 underwent p53 assessment, 272 (55%) were positive, and 114 (42%) were randomly assigned. Accrual was halted on the basis of the data and safety monitoring board review of a futility analysis. Overall 5-year probability of recurring was 0.20 (95% CI, 0.16 to 0.24) with no difference on the basis of p53 status. Only 67% of patients randomly assigned to MVAC received all three cycles with 12 patients receiving no treatment. There was no difference in recurrence in the randomly assigned patients (hazard ratio, 0.78; 95% CI, 0.29 to 2.08; P = .62).
Neither the prognostic value of p53 nor the benefit of MVAC chemotherapy in patients with p53-positive tumors was confirmed, but the high patient refusal rate, lower than expected event rate, and failures to receive assigned therapy severely compromised study power.
Muscle invasive and recurrent nonmuscle invasive bladder cancers have been traditionally treated with a radical cystectomy and urinary diversion. The urinary diversion is generally accomplished through the creation of an incontinent ileal conduit, continent catheterizable reservoir, or orthotopic neobladder utilizing small or large intestine. While radical extirpation of the bladder is often successful from an oncological perspective, there is a significant morbidity associated with enteric interposition within the genitourinary tract. Therefore, there is a great opportunity to decrease the morbidity of the surgical management of bladder cancer through utilization of novel technologies for creating a urinary diversion without the use of intestine. Clinical trials using neourinary conduits (NUC) seeded with autologous smooth muscle cells are currently in progress and may represent a significant surgical advance, potentially eliminating the complications associated with the use of gastrointestinal segments in the urinary reconstruction, simplifying the surgical procedure, and greatly facilitating recovery from cystectomy.
The use of human embryonic stem cells (hESCs) to repair diseased or injured brain is promising technology with significant humanitarian, societal and economic impact. Parkinson’s disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. The generation of this cell type will fulfill a currently unmet therapeutic need. We report on the isolation and perpetuation of a midbrain-specified self-renewable human neural stem cell line (hNSCs) from hESCs. These hNSCs grew as a monolayer and uniformly expressed the neural precursor markers nestin, vimentin and a radial glial phenotype. We describe a process to direct the differentiation of these hNSCs towards the DA lineage. Glial conditioned media acted synergistically with fibroblastic growth factor and leukemia inhibitory factor to induce the expression of the DA marker, tyrosine hydroxylase (TH), in the hNSC progeny. The glial-derived neurotrophic factor did not fully mimic the effects of conditioned media. The hNSCs expressed the midbrain-specific transcription factors Nurr1 and Pitx3. The inductive effects did not modify the level of the glutamic acid decarboxylase (GAD) transcript, a marker for GABAergic neurons, while the TH transcript increased 10-fold. Immunocytochemical analysis demonstrated that the TH-expressing cells did not co-localize with GAD. The transplantation of these DA-induced hNSCs into the non-human primate MPTP model of PD demonstrated that the cells maintain their DA-induced phenotype, extend neurite outgrowths and express synaptic markers.
Background and Purpose
The inflammatory response is a critical component of ischemic stroke. In addition to its physiological role, the mechanisms behind transendothelial recruitment of immune cells also offer a unique therapeutic opportunity for translational stem cell therapies. Recent reports have demonstrated homing of neural stem cells (NSC) into the injured brain areas after intravascular delivery. However, the mechanisms underlying the process of transendothelial recruitment remain largely unknown. Here we describe the critical role of the chemokine CCL2 and its receptor CCR2 in targeted homing of NSC after ischemia.
Twenty-four hours after induction of stroke using the hypoxia-ischemia model in mice CCR2+/+ and CCR2−/− reporter NSC were intra-arterially delivered. Histology and bioluminescence imaging were used to investigate NSC homing to the ischemic brain. Functional outcome was assessed with the horizontal ladder test.
Using NSC isolated from CCR2+/+ and CCR2−/− mice, we show that receptor deficiency significantly impaired transendothelial diapedesis specifically in response to CCL2. Accordingly, wild-type NSC injected into CCL2−/− mice exhibited significantly decreased homing. Bioluminescence imaging showed robust recruitment of CCR2+/+ cells within 6 hours after transplantation in contrast to CCR2−/− cells. Mice receiving CCR2+/+ grafts after ischemic injury showed a significantly improved recovery of neurological deficits as compared to animals with transplantation of CCR2−/− NSC.
The CCL2/CCR2 interaction is critical for transendothelial recruitment of intravascularly delivered NSC in response to ischemic injury. This finding could have significant implications in advancing minimally invasive intravascular therapeutics for regenerative medicine or cell-based drug delivery systems for central nervous system diseases.
chemokines; intravascular transplantation; neural stem cells; regenerative medicine; stroke; transendothelial recruitment
Lithium is a mood stabilizer shown to have neuroprotective effects against several chronic and acute neuronal injuries, including stroke. However, it is unknown whether lithium treatment protects against brain injury post-stroke in a rat model of permanent distal middle cerebral artery occlusion (MCAo) combined with transient bilateral common carotid artery occlusion (CCAo), a model that mimics human stroke with partial reperfusion. In addition, whether lithium treatment alters Akt activity as measured by the kinase activity assay has not been reported, although it is known to inhibit GSK3β activity. After stroke, Akt activity contributes to neuronal survival while GSK3β activity causes neuronal death. We report that a bolus of lithium injection at stroke onset robustly reduced infarct size measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 48 h post-stroke and inhibited cell death in the ischemic penumbra, but not in the ischemic core, as shown by TUNEL staining performed 24 h post-stroke. However, lithium treatment did not alter the reduction in Akt activity as measured by Akt kinase assay. We further showed that lithium did not alter phosphorylated GSK3β protein levels, or the degradation of β-catenin, a substrate of GSK3β, which is consistent with previous findings that long-term treatment is required for lithium to alter GSK3β phosphorylation. In summary, we show innovative data that lithium protects against stroke in a focal ischemia model with partial reperfusion, however, our results dispute the importance of Akt activity in the protective effects of lithium.
Lithium; Akt; Cerebral focal ischemia; GSK3β; β-catenin
Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.
Stroke remains the leading cause of disability in the Western world. Despite decades of work, no clinically effective therapies exist to facilitate recovery from stroke. Stem cells may have the potential to minimize injury and promote recovery after stroke.
Transplanted stem cells have been shown in animal models to migrate to the injured region, secrete neurotrophic compounds, promote revascularization, enhance plasticity and regulate the inflammatory response, thereby minimizing injury. Endogenous neural stem cells also have a remarkable propensity to respond to injury. Under select conditions, subventricular zone progenitors may be mobilized to replace lost neurons. In response to focal infarcts, neuroblasts play important trophic roles to minimize neural injury. Importantly, these endogenous repair mechanisms may be experimentally augmented, leading to robust improvements in function. Ongoing clinical studies are now assessing the safety and feasibility of cell-based therapies for stroke.
We outline the unique challenges and potential pitfalls in the clinical translation of stem cell research for stroke. We then detail what we believe to be the specific basic science and clinical strategies needed to overcome these challenges, fill remaining gaps in knowledge and facilitate development of clinically viable stem cell-based therapies for stroke.
stem cell; neurogenesis; neuroregeneration; stroke; ischemic brain injury; neural progenitor cell; neuroblast; neuroprotection; clinical trial; translational research; plasticity; subventricular zone; migration; differentiation
Gene therapy has demonstrated the protective potential of a variety of genes against stroke. However, conventional gene therapy vectors are limited due to the inability to temporally control their expression, which can sometimes lead to deleterious side effects. Thus, an inducible vector that can be temporally controlled and activated by the insult itself would be advantageous. Using hypoxia responsive elements (HRE) and antioxidant responsive elements (ARE), we have constructed an insult-inducible vector activated by hypoxia and reactive oxygen species (ROS). In COS7 cells, the inducible ARE−HRE-luciferase vectors are highly activated by oxygen deprivation, hydrogen peroxide treatment, and the ROS-induced transcription factor NF-E2-related factor 2 (Nrf2). Using a defective herpes virus, the neuroprotective potential of this inducible vector was tested by over-expressing the transcription factor Nrf2. In primary cortical cultures, expression of the inducible ARE−HRE–Nrf2 protects against oxygen glucose deprivation, similar to that afforded by the constitutively expressed Nrf2. This ARE+HRE vector system is advantageous in that it allows the expression of a transgene to be activated not only during hypoxia but also maintained after reperfusion, thus prolonging the transgene expression during an ischemic insult. This insult-inducible vector system will be a valuable gene therapy tool for activating therapeutic/protective genes in cerebrovascular diseases.
Insult-inducible; Gene therapy; Hypoxia; HIF1; Nrf2; Reactive oxygen species
Moyamoya disease is characterized by the progressive stenosis and often occlusion of the terminal internal carotid arteries, which leads to ischemic and hemorrhagic injuries. The etiology is unknown and surgical revascularization remains the mainstay treatment. We analyzed various hemodynamic factors in 292 patients with moyamoya disease, representing 496 revascularization procedures, including vessel dimension and intraoperative blood flow, using a perivascular ultrasonic flowprobe. Mean middle cerebral artery (MCA) flow rate was 4.4±0.26 mL/min. After superficial temporal artery (STA)–MCA bypass surgery, flows at the microanastomosis were increased fivefold to a mean of 22.2±0.8 mL/min. The MCA flows were significantly lower in the pediatric (16.2±1.3 mL/min) compared with the adult (23.9±1.0 mL/min; P<0.0001) population. Increased local flow rates were associated with clinical improvement. Permanent postoperative complications were low (<5%), but very high postanastomosis MCA flow was associated with postoperative stroke (31.2±6.8 mL/min; P=0.045), hemorrhage (32.1±10.2 mL/min; P=0.045), and transient neurologic deficits (28.6±5.6 mL/min; P=0.047) compared with controls. Other flow and vessel dimension data are presented to elucidate the hemodynamic changes related to the vasculopathy and subsequent to surgical intervention.
cerebral blood flow; extracranial to intracrania bypass; hemodynamic; moyamoya; STA–MCA; vessel diameter
We recently showed that intraischemic moderate hypothermia (30°C) reduces ischemic damage through the Akt pathway after permanent distal middle cerebral artery occlusion in rats. The only Akt pathway component preserved by hypothermia is phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN), which suggests that p-PTEN may have a central role in neuroprotection. Reactive oxygen species (ROS) are critically involved in mediating ischemic damage after stroke by interacting with signaling molecules, including Akt, PTEN, and δ-protein kinase C (PKC). We investigated the protective mechanisms of moderate hypothermia on these signaling proteins after transient focal ischemia in rats. Early moderate hypothermia (3 h) was administered 15 mins before reperfusion, and delayed moderate hypothermia (3 h) was applied 15 mins after reperfusion. Our results indicate that early hypothermia reduced infarction, whereas delayed hypothermia did not. However, both early and delayed hypothermia maintained levels of Mn-SOD (superoxide dismutase) and phosphorylated Akt and blocked δ-PKC cleavage, suggesting that these factors may not be critical to the protection of hypothermia. Nevertheless, early hypothermia preserved p-PTEN levels after reperfusion, whereas delayed hypothermia did not. Furthermore, ROS inhibition maintained levels of p-PTEN after stroke. Together, these findings suggest that phosphorylation levels of PTEN are closely associated with the protective effect of early hypothermia against stroke.
focal ischemia; hypothermia; neuroprotection; stroke
Although the protective mechanisms of delayed ischemic preconditioning have received extensive studies, few have addressed the mechanisms associated with rapid ischemic postconditioning. We investigated whether ischemic tolerance induced by rapid preconditioning is regulated by the Akt survival signaling pathway. Stroke was generated by permanent occlusion of the left distal middle cerebral artery (MCA) plus 30 min or 1 h occlusion of the bilateral common carotid artery (CCA) in male rats. Rapid preconditioning performed 1h before stroke onset reduced infarct size by 69% in rats with 30 min CCA occlusion, but by only 19% with 1 h occlusion. After control ischemia with 30 min CCA occlusion, Western Blot showed that P-Akt was transiently increased while Akt kinase assay showed that Akt activity was decreased. Although preconditioning did not change P-Akt levels at 1h and 5h compared with control ischemia, it attenuated reduction in Akt activity at 5h in the penumbra. However, preconditioning did not change the levels of P-PDK1, P-PTEN, and P-GSK3β in the Akt pathway, all of which were decreased after stroke. At last, the PI3K kinase inhibitor, LY294002, completely reversed the protection from ischemic preconditioning. In conclusion, Akt contributes to the protection of rapid preconditionin against stroke.
rapid preconditioning; ischemic tolerance; cerebral ischemia; focal ischemia; neuroprotection; Akt
Although many studies have shown the great potential of induced hypothermia in stroke treatment, we recognize that there are limitations to the protective effects of hypothermia even in the laboratory. Here, we review our experiments on the protective effects of mild-to-moderate hypothermia in rats. Focal ischemia was induced by bilateral common carotid artery (CCA) occlusion for 1 to 2 hours combined with permanent or transient middle cerebral artery (MCA) occlusion. We compared the effects of mild (33°C) and moderate (30°C) hypothermia, evaluated therapeutic time windows, and studied the underlying mechanisms. On review, our findings revealed that the protective effects of induced mild hypothermia (33°C) were limited, and the therapeutic time window of even moderate hypothermia (30°C) was very short in our specific models, although this limitation might be due to the relatively brief periods of hypothermia used. In addition, we found that hypothermia reduced brain injury by preserving Akt activity, PTEN phosphorylation and εPKC activity, while inhibiting ROS production, and δPKC activity.
Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.
stroke; transplantation; brain rewiring; APP; dendrites
Stem cell transplantation has evolved as a promising experimental treatment approach for stroke. In this review, we address the major hurdles for successful translation from basic research into clinical applications and discuss possible strategies to overcome these issues. We summarize the results from present pre-clinical and clinical studies and focus on specific areas of current controversy and research: (i) the therapeutic time window for cell transplantation; (ii) the selection of patients likely to benefit from such a therapy; (iii) the optimal route of cell delivery to the ischemic brain; (iv) the most suitable cell types and sources; (v) the potential mechanisms of functional recovery after cell transplantation; and (vi) the development of imaging techniques to monitor cell therapy.
Stroke; stem cells; transplantation; regenerative medicine; translational studies
Moyamoya disease is characterized by a chronic stenoocclusive vasculopathy affecting the terminal internal carotid arteries. The clinical presentation and outcome of moyamoya disease remain varied based on angiographic studies alone, and much work has been done to study cerebral hemodynamics in this group of patients. The ability to measure cerebral blood flow (CBF) accurately continues to improve with time, and with it a better understanding of the pathophysiological mechanisms in patients with moyamoya disease. The main imaging techniques used to evaluate cerebral hemodynamics include PET, SPECT, xenon-enhanced CT, dynamic perfusion CT, MR imaging with dynamic susceptibility contrast and with arterial spin labeling, and Doppler ultrasonography. More invasive techniques include intraoperative ultrasonography. The authors review the current knowledge of CBF in this group of patients and the role each main quantitative method has played in evaluating them, both in the disease state and after surgical intervention.
moyamoya disease; cerebral blood flow; hemodynamics; xenon study; positron emission tomography; single-photon emission computed tomography; perfusion imaging
Cerebral cavernous malformations (CCMs) are vascular anomalies of the central nervous system, comprising dilated blood-filled capillaries lacking structural support. The lesions are prone to rupture, resulting in seizures or hemorrhagic stroke. CCM can occur sporadically, manifesting as solitary lesions, but also in families, where multiple lesions generally occur. Familial cases follow autosomal-dominant inheritance due to mutations in one of three genes, CCM1/KRIT1, CCM2/malcavernin or CCM3/PDCD10. The difference in lesion burden between familial and sporadic CCM, combined with limited molecular data, suggests that CCM pathogenesis may follow a two-hit molecular mechanism, similar to that seen for tumor suppressor genes. In this study, we investigate the two-hit hypothesis for CCM pathogenesis. Through repeated cycles of amplification, subcloning and sequencing of multiple clones per amplicon, we identify somatic mutations that are otherwise invisible by direct sequencing of the bulk amplicon. Biallelic germline and somatic mutations were identified in CCM lesions from all three forms of inherited CCMs. The somatic mutations are found only in a subset of the endothelial cells lining the cavernous vessels and not in interstitial lesion cells. These data suggest that CCM lesion genesis requires complete loss of function for one of the CCM genes. Although widely expressed in the different cell types of the brain, these data also suggest a unique role for the CCM proteins in endothelial cell biology.
Two pathways that have been shown to mediate cerebral ischemic damage are the MEK/ERK cascade and the pro-apoptotic δPKC pathway. We investigated the relationship between these pathways in a rat model of focal ischemia by observing and modifying the activation state of each pathway. The ERK1/2 inhibitor, U0126, injected at ischemia onset, attenuated the increase in phosphorylated ERK1/2 (P-ERK1/2) after reperfusion. The δPKC inhibitor, δV1-1, delivered at reperfusion, did not significantly change P-ERK1/2 levels. In contrast, the δPKC activator, ψδRACK, injected at reperfusion, reduced ERK1/2 phosphorylation measured 4 h after reperfusion. Additionally, U0126 pretreatment at ischemia onset reduced infarct size compared with vehicle, but U0126 injected at the onset of reperfusion had no protection. Finally, combination of U0126 injection at ischemia onset plus δV1-1 injection at reperfusion further reduced infarct size, while combination of U0126 delivered at ischemia onset with ψδRACK injected at reperfusion increased infarct size compared with U0126 alone. In conclusion, we find that inhibiting both the MEK/ERK and the δPKC pathways offers greater protection than either alone, indicating they likely act independently.
Cerebral ischemia; MEK/ERK cascade; δPKC; ERK1/2
We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
Beta-catenin can be cleaved by caspase-3 or degraded by activated glycogen synthase kinase-3β via phosphorylating β-catenin. We tested the hypothesis that β-catenin undergoes degradation after stroke, and its degradation is dependent on caspase activity. Stroke was generated by permanent middle cerebral artery occlusion and 1h of transient bilateral common carotid artery occlusion in rats. Active caspase-3 was expressed in the ischemic cortex from 5 to 48 h after stroke, whereas β-catenin markedly degraded at 24 and 48 h after stroke. The caspase 3-specific inhibitor, Z-DQMD-FMK, attenuated β-catenin degradation, but it did not affect phosphorylation of both β-catenin and glycogen synthase kinase-3β. In conclusion, β-catenin degraded after stroke, and its degradation was caspase-3 dependent.
β-catenin; caspase-3; focal ischemia; glycogen synthase kinase-3β; stroke
Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor mediated pathways, such as that triggered by Fas/FasL. Male Sprague Dawley rats subjected to 2h middle cerebral artery occlusion with 2h intraischemic mild hypothermia (33C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by ~50–60% at 6 and 24h post insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by ~50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.
apoptosis; hypothermia; cerebral ischemia; matrix metalloproteinases; Fas/FasL; stroke
Intracerebral injection of the vasoconstrictor peptide, endothelin-1 (ET-1), has been used as a method to induce focal ischemia in rats. The relative technical simplicity of this model makes it attractive for use in mice. However, the effect of ET-1 on mouse brains has not been firmly established. In this study, we determined the ability of ET-1 to induce focal cerebral ischemia in four different mouse strains (CD1, C57/BL6, NOD/SCID, and FVB). In contrast to rats, intracerebral injection of ET-1 did not produce a lesion in any mouse strain tested. A combination of ET-1 injection with either CCA occlusion or N, G-nitro-L-arginine methyl ester (L-NAME) injection produced only a small infarct and its size was strain-dependent. A triple combination of CCA occlusion with co-injection of ET-1 and L-NAME produced a lesion in all mouse strains tested, and this resulted in a significant motor deficit. However, lesion size was still relatively small and strain-dependent. This study shows that ET-1 has a much less potent effect for producing an infarct in mice than rats.
Endothelin-1; focal ischemia; mouse; receptor; endothelial nitric oxide