Artery Occlusion In Adult Rats Research Articles

Endogenous Extracellular Vesicles Participate in Brain Remodeling after Ischemic Stroke

Brain remodeling after an ischemic stroke represents a promising avenue for exploring the cellular mechanisms of endogenous brain repair. A deeper understanding of these mechanisms is crucial for optimizing the safety and efficacy of neuroprotective treatments for stroke patients. Here, we interrogated the role of extracellular vesicles, particularly exosomes, as potential mediators of endogenous repair within the neurovascular unit (NVU). We hypothesized that these extracellular vesicles may play a role in achieving transient stroke neuroprotection. Using the established ischemic stroke model of middle cerebral artery occlusion in adult rats, we detected a surged in the extracellular vesicle marker CD63 in the peri-infarct area that either juxtaposed or co-localized with GFAP-positive glial cells, MAP2-labeled young neurons, and VEGF-marked angiogenic cells. This novel observation that CD63 exosomes spatially and temporally approximated glial activation, neurogenesis, and angiogenesis suggests that extracellular vesicles, especially exosomes, contribute to the endogenous repair of the NVU, warranting exploration of extracellular vesicle-based stroke therapeutics.

Eyeballing stroke: Blood flow alterations in the eye and visual impairments following transient middle cerebral artery occlusion in adult rats

Middle cerebral artery occlusion in rodents remains a widely used model of ischemic stroke. Recently, we reported the occurrence of retinal ischemia in animals subjected to middle cerebral artery occlusion, owing in part to the circulatory juxtaposition of the ophthalmic artery to the middle cerebral artery. In this study, we examined the eye hemodynamics and visual deficits in middle cerebral artery occlusion-induced stroke rats. The brain and eye were evaluated by laser Doppler at baseline (prior to middle cerebral artery occlusion), during and after middle cerebral artery occlusion. Retinal function-relevant behavioral and histological outcomes were performed at 3 and 14 days post-middle cerebral artery occlusion. Laser Doppler revealed a typical reduction of at least 80% in the ipsilateral frontoparietal cortical area of the brain during middle cerebral artery occlusion compared to baseline, which returned to near-baseline levels during reperfusion. Retinal perfusion defects closely paralleled the timing of cerebral blood flow alterations in the acute stages of middle cerebral artery occlusion in adult rats, characterized by a significant blood flow defect in the ipsilateral eye with at least 90% reduction during middle cerebral artery occlusion compared to baseline, which was restored to near-baseline levels during reperfusion. Moreover, retinal ganglion cell density and optic nerve depth were significantly decreased in the ipsilateral eye. In addition, the stroke rats displayed eye closure. Behavioral performance in a light stimulus-mediated avoidance test was significantly impaired in middle cerebral artery occlusion rats compared to control animals. In view of visual deficits in stroke patients, closely monitoring of brain and retinal perfusion via laser Doppler measurements and examination of visual impairments may facilitate the diagnosis and the treatment of stroke, including retinal ischemia.

Delayed epidural transplantation of human induced pluripotent stem cell-derived neural progenitors enhances functional recovery after stroke

Induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) are a promising source of tailor-made cell therapy for neurological diseases. However, major obstacles to clinical use still exist. To circumvent complications related to intracerebral administration, we implanted human iPSC-NPCs epidurally over the peri-infarct cortex 7 days after permanent middle cerebral artery occlusion in adult rats. Compared to controls, cell-treated rats showed significant improvements in paretic forelimb usage and grip strength from 10 days post-transplantation (dpt) onwards, as well as reductions in lesion volumes, inflammatory infiltration and astrogliosis at 21 dpt. Few iPSC-NPCs migrated into rat peri-infarct cortices and exhibited poor survival in tissue. To examine the paracrine therapeutic mechanisms of epidural iPSC-NPC grafts, we used transmembrane co-cultures of human iPSC-NPCs with rat cortical cells subjected to oxygen-glucose deprivation. Compared to other human stem cells, iPSC-NPCs were superior at promoting neuronal survival and outgrowth, and mitigating astrogliosis. Using comparative whole-genome microarrays and cytokine neutralization, we identified a neurorestorative secretome from iPSC-NPCs, and neutralizing enriched cytokines abolished neuroprotective effects in co-cultures. This proof-of-concept study demonstrates a relatively safe, yet effective epidural route for delivering human iPSC-NPCs, which acts predominately through discrete paracrine effects to promote functional recovery after stroke.

Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro

Carthamus tinctorius L. (safflower) is one of the most commonly used Chinese herbal medicines to prevent and treat cardiac disease in clinical practice. However, the mechanisms responsible for such protective effects remain largely unknown. In this study, we investigated the anti-myocardial ischemia effects of a purified extract of C. tinctorius (ECT) both in vivo and in vitro. An animal model of myocardial ischemia injury was induced by left anterior descending coronary artery occlusion in adult rats. Pretreatment with ECT (100, 200, 400, 600 mg/kg body wt.) could protect the heart from ischemia injury by limiting infarct size and improving cardiac function. In the in vitro experiment, neonatal rat ventricular myocytes were incubated to test the direct cytoprotective effect of ECT against H(2)O(2) exposure. Pretreatment with 100-400 microg/ml ECT prior to H(2)O(2) exposure significantly increased cell viability as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ECT also markedly attenuated H(2)O(2)-induced cardiomyocyte apoptosis, as detected by Annexin V and PI double labeling with flow cytometry. The intracellular level of reactive oxygen species (ROS) was shown by 2',7'-dichlorofluorescin diacetate (DCFH-DA), and ECT pretreatment significantly inhibited H(2)O(2)-induced ROS increase. We made a preliminary examination of the signaling cascade involved in ECT mediated anti-apoptotic effects. Phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) blocked the cytoprotective effect conferred by ECT. Taken together, our findings provide the first evidence that the cardioprotective effects of ECT in myocardial ischemia operate partially through reducing oxidative stress induced damage and apoptosis. The protection is achieved by scavenging of ROS and mediating the PI3K signaling pathway.

Blood micromolar concentrations of kaempferol afford protection against ischemia/reperfusion-induced damage in rat brain

The slow time course of neurodegeneration after brain ischemia/reperfusion opened a realistic time window for the application of protective therapies to prevent spreading of brain damage. In this work, we studied the ability of micromolar concentrations of this flavonoid in the blood to protect against brain damage induced by transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in adult rats and brain damage has been monitored by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H-E) staining, 'in situ' terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL), 'in situ' metalloproteinase activity using DQ-gelatin and loss of anti-laminin staining. Intravenous injections of kaempferol, at a dose of 10-15 mumol/L of blood 30 min before the induction of a 60 min ischemia-episode and just after reperfusion, led to >90% and 70-80% (TTC, H-E, TUNEL) decrease of brain damage in the temporal-frontal areas of neocortex and striatum, respectively, but only 40-50% decrease of brain damage was observed in the hippocampus and vicinal caudal areas of the striatum. This treatment with kaempferol also produced a similar reduction of metalloproteinase activation and loss of anti-laminin staining in cortical and striatum infarct areas. Kaempferol treatment efficiently protected against nitrosative-oxidative stress after ischemia/reperfusion, as shown by nearly complete protection against the increase of protein nitrotyrosines, and also afforded strong protection against the increase of apoptotic cell death (TUNEL) and biochemical markers of apoptosis, such as caspase-9 activity and poly-(ADP-ribose) polymerase degradation. On these grounds, a potential new therapeutic role of kaempferol to acute treatment of ischemic stroke is suggested.

Intrathecal treatment with anti-Nogo-A antibody improves functional recovery in adult rats after stroke

Stroke often results in devastating neurological disabilities with no specific treatment available to improve functional recovery. Neurite growth inhibitory proteins such as Nogo-A play a critical role in impeding regain of function after stroke. We have reported that treatment with anti-Nogo-A antibody using the intracerebroventricular route resulted in improvement of function and neuroplasticity in adult or aged rats after stroke. This present study tested a more clinically accessible route for applying anti-Nogo-A antibodies, the intrathecal route. Anti-Nogo-A or control antibody was administered intrathecally at lower lumbar levels 1 week after middle cerebral artery occlusion in adult rats. Our results show that anti-Nogo-A antibody delivered by this intrathecal route for 2 weeks penetrated into brain parenchyma and bound to myelin-enriched structures such as the corpus callosum and striatal white matter. Animals receiving anti-Nogo-A antibody treatment significantly improved recovery of function on the skilled forelimb reaching task as compared to stroke only and stroke/control antibody animals. These findings show that anti-Nogo-A antibody delivered through the intrathecal route is as effective in restoring lost functions after stroke as the intracerebroventricular route. This is of great importance for the future application of anti-Nogo-A immunotherapy for ischemic stroke treatment.

JAK2 and STAT3 activation contributes to neuronal damage following transient focal cerebral ischemia

Increased levels of interleukin-6 (IL-6) play a role in post-ischemic cerebral inflammation. IL-6 binding to its receptors induces phosphorylation of the receptor associated janus kinases (JAKs), and the down-stream signal transducer and activator of transcription (STAT) family of transcription factors, which amplify the IL-6 signal transduction. We evaluated the functional significance of JAK2 and STAT3 activation in focal ischemia-induced neuronal damage. Transient middle cerebral artery occlusion in adult rats led to increased JAK2 and STAT3 phosphorylation in the ipsilateral cortex and striatum after 6-72 h of reperfusion. Fluorescent immunohistochemistry with cell specific markers (NeuN for neurons, glial fibrillary acidic protein for reactive astrocytes and ED1/OX42 for activated macrophages/microglia) showed that both pJAK2 and pSTAT3 staining is predominantly localized in the macrophages/microglia in the post-ischemic brain. Intracerebroventricular infusion of rats with AG490 (a JAK2 phosphorylation inhibitor) prevented the post-ischemic JAK2 and STAT3 phosphorylation and significantly decreased the infarct volume, number of apoptotic cells and neurological deficits, compared to vehicle control. Furthermore, intracerebral injection of siRNA specific for STAT3 led to curtailed STAT3 mRNA expression and phosphorylation, decreased infarct volume, fewer apoptotic cells and improved neurological function following transient middle cerebral artery occlusion. These studies show that JAK2-STAT3 activation plays a role in post-ischemic brain damage.

Neuroprotection by pyrroloquinoline quinone (PQQ) in reversible middle cerebral artery occlusion in the adult rat

Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and redox modulator. It has previously been reported to reduce infarct size in 7-day-old rat pups with an in vivo cerebral hypoxia/ischemia model (Jensen et al., 1994). In this study, we tested whether improvement is found in both behavioral measures of protection and by histological measures of infarcted tissue at 72 h after reversible middle cerebral artery occlusion (rMCAo) in adult rats. Two-hour rMCAo was induced in adult rats using the intraluminal suture technique. PQQ (10, 3, and 1 mg/kg) was given once by intravenous injection at the initiation, or 3 h after the initiation, of 2 h rMCAo. Neurobehavioral deficits were evaluated daily for 3 days followed by infarct volumes measurements by 2,3,5-triphenyltetrazolium chloride (TTC) staining. PQQ at 10 mg/kg infused at the initiation, or 3 h after the initiation, of rMCAo was effective in reducing cerebral infarct volumes measured 72 h later. At 3 h after ischemia, a dose of 3 mg/kg significantly reduced infarct volume compared to vehicle-treated animals, but 1 mg/kg was ineffective. Neurobehavioral scores were also significantly better in the PQQ-treated group compared to the vehicle controls when PQQ was given at 10 and 3 mg/kg, but not at 1 mg/kg. Thus, PQQ is neuroprotective when given as a single administration at least 3 h after initiation of rMCAo. These data indicate that PQQ may be a useful neuroprotectant in stroke therapy.

CRP or not CRP? That Is the Question

C-reactive protein (CRP) and cardiovascular disease is a very hot topic at present. Excitement and interest have spilled over dramatically from the scientific literature into the media and popular press leading to much speculative comment. However, rigorously controlled and reproducible studies are now laying the basis for a more realistic consensus. The article in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology from Carmen van den Berg’s laboratory makes a notable contribution to this process. 1 See page 1225 Shortly after its discovery in 1929 as “the acute phase protein,” increased production of CRP was recognized as a characteristic feature of the response to acute myocardial infarction, and until the 1960’s detection of CRP was widely used for routine monitoring of acute rheumatic fever. In 1961 Irving Kushner, in one of the first applications of the then new technique of immunofluorescence, demonstrated the deposition of rabbit CRP in experimental myocardial infarction lesions. The association between CRP and cardiovascular disease thus has a very long history. In 1979 Kushner reported the kinetics of the acute phase CRP response to human acute myocardial infarction, and soon afterward we first investigated critically the behavior of CRP in clinical coronary artery disease and myocardial infarction.2 We observed that persistently elevated circulating CRP concentrations after an infarct were associated with a poor prognosis, although at that time we were focusing mainly on CRP as a sensitive nonspecific marker of all the various complications of coronary occlusion and their treatment. The current phase of interest in CRP and cardiovascular disease started in the early 1990’s with observations of increased CRP concentrations in some patients with “active coronary syndromes” and some individuals with acute myocardial infarction tested very soon after onset of pain, before the acute phase response to infarction could have started. These findings were consistent with the growing recognition at that time of the importance of inflammation in atherogenesis and the inflammatory nature of unstable atherosclerotic lesions. At the same time the large European prospective study (ECAT) of coagulation factors as possible prognostic markers in outpatients with stable and unstable angina unexpectedly revealed that the baseline value of CRP, which had only been included as a control for the acute phase behavior of some clotting proteins, significantly predicted future coronary events.3 The assay used was insufficiently sensitive to detect CRP in many of the samples in the ECAT study, and we therefore developed the first automated high-sensitivity method to re-assay their 3000 samples. Separately we also used this method to study, in collaboration with the Maseri group, carefully characterized patients with severe unstable angina who had not yet experienced any myocardial necrosis. We selected a cut point of 3 mg/L as the upper limit of normal, based on our original 1981 study of 468 healthy volunteer blood donors in whom this was the 90th centile of the CRP distribution. 4 In both the ECAT and the unstable angina studies, increased baseline CRP values were associated with significantly increased risk of future coronary events. 5,6 Subsequently several groups showed independently that baseline CRP measurements are associated with future coronary events in general populations, without known preexisting coronary artery disease. 7–10 In 2000 we reported with Danesh and colleagues the results from the British Regional Heart Study comprising 506 coronary events, 2 as many as in any other such study up to that time, together with a meta-analysis of all previous studies.11 This showed that the relative risk of having a coronary event among individuals with a baseline CRP value in the upper compared with the lower tertile of the CRP distribution was 2.0. In 2004 the Reykjavik Study, comprising 2459 coronary events, showed this relative risk to be 1.45 (95% CI, 1.25 to 168), and meta-analysis of all previously published general populations studies, comprising 7068 patients with coronary events, gave a similar result. 12 The results from this now quite rigorously

Human C-Reactive Protein Increases Cerebral Infarct Size after Middle Cerebral Artery Occlusion in Adult Rats

Human C-reactive protein (CRP), the classic acute phase plasma protein, increases in concentration after myocardial infarction and stroke. Human CRP binds to ligands exposed in damaged tissue and can then activate complement and its proinflammatory functions. In contrast, rat CRP, which binds to similar ligands, does not activate complement. In the present study, systemic complement depletion with cobra venom factor in adult rats subjected to middle cerebral artery occlusion did not affect cerebral infarct size, indicating that circulating complement does not contribute to injury in this model. However, we have previously reported that administration of human CRP to rats undergoing coronary artery ligation caused a marked increase in size of the resulting myocardial infarction, associated with codeposition of human CRP and rat complement in the infarcts. In the present study, we show that adult rats subjected to middle cerebral artery occlusion and then treated with human CRP similarly developed significantly larger cerebral infarcts compared with control subjects receiving human serum albumin. Human CRP can thus contribute to ischemic tissue damage in the brain as well as in the heart, and inhibition of CRP binding may therefore be a promising target for tissue protective acute therapeutic intervention in stroke as well as in myocardial infarction.

Neuronal replacement from endogenous precursors in the adult brain after stroke.

In the adult brain, new neurons are continuously generated in the subventricular zone and dentate gyrus, but it is unknown whether these neurons can replace those lost following damage or disease. Here we show that stroke, caused by transient middle cerebral artery occlusion in adult rats, leads to a marked increase of cell proliferation in the subventricular zone. Stroke-generated new neurons, as well as neuroblasts probably already formed before the insult, migrate into the severely damaged area of the striatum, where they express markers of developing and mature, striatal medium-sized spiny neurons. Thus, stroke induces differentiation of new neurons into the phenotype of most of the neurons destroyed by the ischemic lesion. Here we show that the adult brain has the capacity for self-repair after insults causing extensive neuronal death. If the new neurons are functional and their formation can be stimulated, a novel therapeutic strategy might be developed for stroke in humans.

CD95 Ligand (Fas-L/APO-1L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Mediate Ischemia-Induced Apoptosis in Neurons

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Nitric oxide production during focal cerebral ischemia in rats.

Nitric oxide has been implicated as a mediator of glutamate excitotoxicity in primary neuronal cultures. A number of indicators of brain nitric oxide production (nitric and cyclic guanosine monophosphate [cGMP] concentrations and nitric oxide synthase activity) were examined after bilateral carotid ligation and right middle cerebral artery occlusion in adult rats. Brain nitrite was significantly increased in the right versus left cortex 5, 10, and 20 minutes after middle cerebral artery occlusion (P < .05), with a return to baseline at 60 minutes. There were no significant changes in cerebellar concentrations. Cortical levels of cGMP were increased at 10, 20, and 60 minutes after occlusion, with significant right-to-left differences (P < .05). Cerebellar concentrations of cGMP were also increased but without significant side-to-side differences. Nitric oxide synthase activity increased approximately 10-fold from baseline 10 minutes after occlusion in the right cortex but decreased markedly by 60 minutes from its peak at 10 minutes. The right-to-left difference in nitric oxide synthase activity was significant at 20 minutes (P < .05). Pretreatment of rats with NG-nitro-L-arginine, a nitric oxide synthase inhibitor, abolished the rise in nitrite and cGMP. These results suggest that a sharp transient increase in the activity of nitric oxide synthase occurs during the first hour of cerebral ischemia, which leads to a burst in nitric oxide production and activation of guanylate cyclase.

Effect of dimethyl sulfoxide and glycerol on acute bowel ischemia in the rat

After a 30 minute period of superior mesenteric artery occlusion in adult rats, there was a significant decrease in peritoneal inflammatory reaction, ileus, peritoneal adhesion formation, and histologically proved bowel wall necrosis in animals given intravenous dimethyl sulfoxide at the end of the ischemic period. In contrast, control rats given normal saline solution intravenously demonstrated severe inflammatory reaction, ileus, hemorrhagic peritoneal fluid, extensive adhesion formation, and areas of bowel wall necrosis. Intravenous glycerol did not have the beneficial effect seen with dimethyl sulfoxide. Neither compound was effective when given intraperitoneally. We conclude that intravenous dimethyl sulfoxide has a significant protective effect in rats with acute intestinal ischemia due to the superior mesenteric artery occlusion in the rat.