Bilateral Carotid Artery Ligation Research Articles

Experimental rat model of chronic cerebral hypoperfusion-reperfusion mimicking normal perfusion pressure breakthrough phenomenon.

Normal perfusion pressure breakthrough (NPPB) phenomenon is a major life-threatening complication that restricts the treatment of complex intracranial arteriovenous malformations. The aim of the study it to develop a rat model mimicking NPPB phenomenon that enables the evaluation of any therapy to prevent such complication. Twenty Wistar male rats were randomly assigned to either a study or a control group. Study animals underwent an end-to-side left external jugular vein-common carotid artery anastomosis and ligation of bilateral external carotid arteries. Control animals only underwent ligation of bilateral external carotid arteries. All animals were sacrificed sixty days after the procedure. Hemodynamic parameters [mean arterial pressure (MAP), intracranial pressure (ICP) and cerebral perfusion pressure (CPP)], blood-brain barrier (BBB) permeability (measured by fluorescein staining) and histological features were then compared between both groups. A significant decrease in MAP and CPP was confirmed in the study group. An increase in ICP was also observed. A significant decrease in MAP and CPP was also present in the study group when comparing preoperative values with those recorded on days 0 (postoperative), 7 and 60. Fluorescein staining findings were consistent with signs of BBB disruption in study animals. Histological analysis demonstrated an increased number of pyknotic neurons in the ipsilateral hemisphere of rat brains included in the study group. These results confirm that this model mimics a vascular steal state with chronic cerebral hypoperfusion comparable to patients with AVMs behavior and disruption of the BBB after fistula closure comparable to NPPB phenomenon disorders.

Microneurosurgical techniques and perioperative strategies utilized to optimize experimental supracollicular decerebration in rats.

Decerebration permits neurophysiological experimentation absent the confounding effects of anesthesia. Use of the unanesthetized decerebrate preparation in vivo offers several advantages compared with recordings performed in reduced slice preparations, providing the capacity to perform extracellular and intracellular neuronal recordings in the presence of an intact brainstem network. The decerebration procedure typically generates variable degrees of blood loss, which often compromises the hemodynamic stability of the preparation. We describe our microsurgical techniques and discuss microsurgical pearls utilized in order to consistently generate normotensive supracollicularly decerebrate preparations of the rat, exhibiting an augmenting pattern of phrenic nerve discharge. In brief, we perform bilateral ligation of the internal carotid arteries, biparietal craniectomies, securing of the superior sagittal sinus to the overlying strip of bone, removal of the median strip of bone overlying the superior sagittal sinus, supracollicular decerebrative encephalotomy, removal of the cerebral hemispheres, and packing of the anterior and middle cranial fossae with thrombin soaked gelfoam sponges. Hypothermia and potent inhalational anesthesia ensure neuroprotection during postdecerebrative neurogenic shock. Advantages of our approach include a bloodless and fast operation with a nil percent rate of operative mortality. We allow animal arterial pressure to recover gradually in parallel with gentle weaning of anesthesia following decerebration, performed contemporaneously with the provision of the neuromuscular antagonist vecuronium. Anesthetic weaning and institution of vecuronium should be contemporaneous, coordinate, gentle, gradual, and guided by the spontaneous recovery of the arterial blood pressure. We describe our microsurgical techniques and perioperative management strategy designed to achieve decerebration and accordingly survey the literature on techniques used across several studies in achieving these goals.

Morphological features of parietal cortex and hippocampus neuron of rats following subtotal cerebral ischemia associated with omega-3 polyunsaturated fatty acids injection

Aim of the study was to investigate the morphological features of neurons of the parietal cortex and hippocampus of rats with subtotal cerebral ischemia (SACI) during administration of omega-3 polyunsaturated fatty acids (omega-3 PUFA). Materials and methods . The experiments were performed on 24 white outbred male rats. Surgical interventions were carried out under conditions of intravenous thiopental anesthesia (40–50 mg/kg). Acute SACI was modeled by bilateral ligation of the common carotid arteries. The animals of the main group ( n = 6, SACI + omega-3 PUFA) received intragastric injections of omega-3 PUFA preparation at a dose of 5 mg/kg body weight prior to ischemia for a week. In the comparison group ( n = 6, SACI), the drug was not used; the control ( n = 6) was sham operated animals, which were incised without skin ligation. Animals were decapitated after 60 minutes of ischemia. In rats, morphological changes in the neurons of the parietal cortex and the CA1 field of the hippocampus were studied. For morphometric and histochemical studies, animals were quickly removed after decapitation of the brain, pieces of the anterior cortex of the cerebral hemispheres were fixed in Carnoy fluid. Serial paraffin sections were stained with 0.1 % toluidine blue according to the Nissl method. Statistical hypothesis testing was performed using the Kruskal-Wallis ANOVA test. Results and discussion . The morphological and functional disorders in the parietal cortex and hippocampus have been revealed in animals of both experimental groups. The appointment of omega-3 did not significantly affect the size and shape of neurons in both the parietal cortex and the hippocampus. In the hippocampus, the administration of omega-3 polyunsaturated fatty acids resulted in a decrease in the number of hyperchromic shriveled neurons (by 20%) and an increase in hyperchromic neurons by 31 %. The number of shadow cells in this section did not differ from the indices of the control group. In the parietal cortex, no corrective effect was noted. Conclusions. Thus, subtotal cerebral ischemia leads to the development of morphofunctional disorders of the cerebral cortex. Administration of omega-3 had a positive effect on the state of rat hippocampal neurons, reducing the number of degenerative forms of neurons. The data obtained can serve as the basis for the search for new approaches to the treatment of acute ischemic stroke, which is one of the urgent problems of experimental and clinical neurology.

Erythropoietin and vascular endothelial growth factor level in normoxia and in cerebral ischemia under pharmacological and hypoxic preconditioning

The level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF-A) was investigated in blood serum and brain of Wistar rats by the enzyme immunoassay with specific rat antibodies. These growth factors are actively studied as biomarkers of ischemia or cytoprotection, as well as targets for agents initiating preconditioning (PreC). Pharmacological (amtizol administration), hypoxic (hypobaric hypoxia), and combined PreC (amtizol+hypobaric hypoxia) were used as neuroprotective approaches in this experimental work. In normoxia groups blood and brain tissue were collected 1 h (early period) or 48 h (delayed period) after the PreC. In addition we studied groups of animals with cerebral ischemia (induced by bilateral ligation of the common carotid arteries) 1 h and 48 h after the combined PreC: the levels of EPO and VEGF-A in the blood serum and the brain supernatant were determined in one day after the ligation. Experiments have shown that amtizol (3,5-diamino-1,2,4-thiadiazole) in normoxia increased the EPO level in the brain, and did not change EPO in blood serum and VEGF-A levels in both serum and the brain. A three-day (60 min exposure with 48 h intervals) hypobaric hypoxia (410 mm Hg) increased EPO and VEGF-A in the blood serum and brain tissues, but in most experimental groups differences did not reach the level of statistical significance versus intact control. The combined PreC was accompanied by a significant increase of EPO and VEGF-A in normoxia conditions both in early and delayed period of PreC. In cerebral ischemia the EPO level in the blood serum and brain tissues was higher than in intact control. The serum level of VEGF-A of the ischemia control group tended to increase while the brain level of VEGF-A remained basically unchanged versus the intact control group. In combined PreC before ischemia, the EPO level was lower in serum as compared with the ischemia control in the delayed PreC period, but did not differ significantly from the ischemia control in serum in early period and in brain tissues in both PreC periods. The VEGF-A level in the groups of combined PreC was significantly lower in serum as compared with the ischemia control in both the early and delayed PreC; in brain tissues it did not differ from the level of both the intact and ishemia control in early PreC period and was higher than in both control groups in the delayed PreC period.

Changes in the Cellular Composition of the Cerebral Cortex in Rats with Different Levels of Cognitive Functions Under Cerebral Hypoperfusion

The aim of study was to investigate characteristics of neurodystrophic changes in neurons and glia of the motor cortex in rats with different levels of cognitive functions under bilateral ligation of the common carotid arteries. Material and methods . The study included 136 Wistar rats. All animals were divided into two subgroups based on the results of the Morris water maze test: with high and low level of ability to spatial learning. Animals of the experimental group were removed from the experiment on the 1st, 6th, 14th, 21st, 35th, 60th and 90th days after bilateral ligation of both carotid arteries. Histological sections of the motor cortex were studied using Nissl and hematoxylin-eosin staining. Results . In 1, 6 and 8 days after ischemia, the number of neurons with irreversible changes and dead cells reached their maximum for the entire time of observation. On the 8th day of study, compact groups of glial cells appeared near the vessels. Heterochrony was noted: in animals with high level of cognitive ability, an increase in the number of neurons with irreversible changes followed the peak of cell death; in animals with low level, on the contrary, the maximum number of dead cells was observed on the 6th day, and the maximum number of neurons with irreversible changes – on the 1st. On the 14th, 21st, and 28th days there was observed a gradual stabilization of indicators characterizing damage to the cerebral cortex. Average values of the perikarya area and neurons nuclei increased, the nuclear-cytoplasmic ratio decreased, and intracellular swelling was noted. After 35 days, areas of the cortex depleted in bodies of neurons and glia appeared; the number of neurons with irreversible changes increased, to a greater extent, in animals with high level of cognitive abilities. Typical trends of the first month of study are as follows: a decrease in the nuclear-cytoplasmic ratio and a number of neurons without irreversible changes, and an increase in the neuroglial index continued to progress on the 60th and 90th days of study. The apical dendrites of the pyramidal neurons obtained a corkscrew course. Compact groups of glial cells disappeared. Conclusion . During the first week of cerebral hypoperfusion, irreversible changes in neurons predominated, in the second and third weeks, the morphological criteria of their functional activity decreased. In the fourth and fifth weeks incomplete adaptation developed in the form of an increased number of neurons near the vessels of the hemocirculatory bed and an increased number of satellite gliocytes by immersing them in the cytoplasm of neurons. In 2-3 months after cerebral hypoperfusion, signs of acute hypoxia reappeared. Animals with high level of cognitive ability were characterized by large damage to the structures of the neuroglial ensemble.

Possible mechanisms of lycopene amelioration of learning and memory impairment in rats with vascular dementia.

Oxidative stress is involved in the pathogenesis of vascular dementia. Studies have shown that lycopene can significantly inhibit oxidative stress; therefore, we hypothesized that lycopene can reduce the level of oxidative stress in vascular dementia. A vascular dementia model was established by permanent bilateral ligation of common carotid arteries. The dosage groups were treated with lycopene (50, 100 and 200 mg/kg) every other day for 2 months. Rats without bilateral carotid artery ligation were prepared as a sham group. To test the ability of learning and memory, the Morris water maze was used to detect the average escape latency and the change of search strategy. Hematoxylin-eosin staining was used to observe changes of hippocampal neurons. The levels of oxidative stress factors, superoxide dismutase and malondialdehyde, were measured in the hippocampus by biochemical detection. The levels of reactive oxygen species in the hippocampus were observed by dihydroethidium staining. The distribution and expression of oxidative stress related protein, neuron-restrictive silencer factor, in hippocampal neurons were detected by immunofluorescence histochemistry and western blot assays. After 2 months of drug administration, (1) in the model group, the average escape latency was longer than that of the sham group, and the proportion of straight and tend tactics was lower than that of the sham group, and the hippocampal neurons were irregularly arranged and the cytoplasm was hyperchromatic. (2) The levels of reactive oxygen species and malondialdehyde in the hippocampus of the model group rats were increased, and the activity of superoxide dismutase was decreased. (3) Lycopene (50, 100 and 200 mg/kg) intervention improved the above changes, and the lycopene 100 mg/kg group showed the most significant improvement effect. (4) Neuron-restrictive silencer factor expression in the hippocampus was lower in the sham group and the lycopene 100 mg/kg group than in the model group. (5) The above data indicate that lycopene 100 mg/kg could protect against the learning-memory ability impairment of vascular dementia rats. The protective mechanism was achieved by inhibiting oxidative stress in the hippocampus. The experiment was approved by the Animal Ethics Committee of Fujian Medical University, China (approval No. 2014-025) in June 2014.

Effect of Different Doses and Times of FK506 on Different Areas of the Hippocampus in the Rat Model of Transient Global Cerebral Ischemia-Reperfusion.

Background Stroke is a major worldwide problem that is leading to a high mortality rate in humans. Ischemia, as the most common type of stroke, is characterized by tissue damage that can occur due to insufficient blood flow to the brain even for a brief duration, leading to the release of inflammatory factors, cytokines, and free radicals. In this study, we investigated the effective dose and injection time of FK506 as an immunophilin ligand for providing a suitable effect on cells of CA2, CA3, and dentate gyrus of the hippocampus. Methods In this in vivo study, a total of 48 male Wistar rats were divided into nine groups. The ischemia model was induced by the ligation of bilateral common carotid arteries. The doses of FK506 (3, 6, and 10 mg/kg) were administered intravenously (IV) at the beginning of reperfusion, followed by repeated injections (10 mg/kg) at 6, 24, 48, and 72 hours after ischemia, respectively. Brains were removed and prepared for Nissl staining and the TdT-mediated dUTP Nick End Labeling method. Results Data showed that global ischemia did not decrease the number of viable pyramidal cells in CA2 and CA3 regions, but significant differences were observed in the number of viable granular cells and apoptotic bodies in the dentate gyrus between the control and ischemia groups. Repeated doses of 6 mg/kg of FK506 at an interval of 48 hours were deemed to be the suitable dose and best time of injection. Conclusions It seems that FK506 can ameliorate the extent of apoptosis and may be a good candidate for the treatment of ischemia-induced brain damage.

Cranial burr hole with erythropoietin administration induces reverse arteriogenesis from the enriched extracranium

It is challenging to revitalize ischemic penumbra after an acute stroke with intracranial perfusion insufficiency. To evaluate whether cranial burr hole and erythropoietin (EPO) generate effective revascularization, we investigated the efficacy of the augmentation method for reverse arteriogenesis from the healthy extracranial milieu. An intracranial perfusion insufficiency was created through bilateral internal carotid artery ligation (bICAL) in Sprague-Dawley rats. We administered recombinant human EPO (5000 U/kg) or saline intraperitoneally for 3 days after bICAL. Mechanical barrier disruption (MBD) was performed through a cranial burr hole with small dural cracks in the right hemisphere. The ipsilateral hemisphere with MBD grossly showed vascular networks between the extra- and intra-cranial spaces 2 weeks after the MBD procedure. It also showed significantly increased vessels in the intracranial vasculature adjacent to the MBD region (p = 0.0006). The levels of pro-angiogenic and inflammatory factors with prominent markers of vessel permeability were also significantly increased (MBD-only vs. control; Tnf-α, p = 0.0007; Vegf, p = 0.0206). In the EPO-administered group, such elevations in inflammation were significantly mitigated (combined vs. MBD-only; Tnf-α, p = 0.0008). The ipsilateral hemisphere with MBD-EPO (vs. MBD-only) showed significantly increased vessels (RECA-1, p = 0.0182) and their maturation (RECA-1/α-SMA, p = 0.0046), with upregulation of tumor growth factor-β1 (Tgf-β1, p = 0.037) and matrix metalloproteinase-2 (Mmp-2, p = 0.0488). These findings were completely blocked by minocycline (MIC) administration during in vivo (Tgf-β1, p = 0.0009; Mmp-2, p < 0.0001) and in vitro experiments (tube formation, p < 0.0001). Our data suggest that the MBD procedure (for angiogenic routes) and EPO administration (for an arteriogenic booster) are complimentary and can facilitate successfully “reverse arteriogenesis” in subjects with intracranial perfusion insufficiency.

Noninvasive noncontact speckle contrast diffuse correlation tomography of cerebral blood flow in rats

Continuous and longitudinal imaging of cerebral blood flow (CBF) variations provide vital information to investigate pathophysiology and interventions for a variety of neurological and cerebral diseases. An innovative noncontact speckle contrast diffuse correlation tomography (scDCT) system was downscaled and adapted for noninvasive imaging of CBF distributions in rat brain through intact scalp and skull. Algorithms for 2D mapping and 3D image reconstruction of CBF distributions were developed and optimized. The continuous imaging capability of the system was shown by imaging global CBF increases during CO2 inhalations and regional CBF decreases across two hemispheres during sequential unilateral and bilateral common carotid artery ligations. The longitudinal imaging capability was demonstrated by imaging CBF variations over a long recovery period of 14 days after an acute stroke. Compared to the 2D mapping method, the 3D imaging method reduces partial volume effects, but needs more computation time for image reconstruction. Results from this study generally agree with those reported in the literature using similar protocols to induce CBF changes in rats. The scDCT enables a relatively large penetration depth (up to ∼10 mm), which is sufficient for transcranial brain measurements in small animals and human neonates. Ultimately, we expect to provide a noninvasive noncontact cerebral imager for basic neuroscience research in small animal models and clinical applications in human neonates.

PATHOLOGICAL EVALUATION OF NEONATAL FERRET MODELS OF INFLAMMATION‐SENSITIZED HYPOXIA‐ISCHEMIA

BackgroundThere is a need for relevant animal models for encephalopathy of prematurity and neonatal encephalopathy, and the ferret, with a gyrified brain and similar white:gray matter ratio to humans, is an appealing potential model species. The P10 ferret is approximately equivalent to a human infant at 23–26 weeks gestation, and the P21 ferret corresponds to approximately 40 weeks human gestation.MethodsFour different protocols to create ferret models of ischemia related preterm and term injury were evaluated: (1) intraperitoneal lipopolysaccharide (LPS; 5 mg/kg) administration at postnatal day (P)11 followed by consecutive hypoxia/hyperoxia/hypoxia (60min at 9%, 120min at 60%, 30min at 9%) in a humidified chamber with a target intra‐hypoxic rectal temperature of 37°C; (2) carotid artery ligation at P11 in combination with LPS administration and hypoxia/hyperoxia/hypoxia; (3) bilateral carotid artery ligation at P21 with LPS administration and hypoxia/hyperoxia/hypoxia (4) bilateral carotid artery ligation at P17 with LPS administration and hypoxia/hyperoxia/hypoxia, followed by 5 hours of prolonged temperature management to prevent anapyrexia. Control animals received saline vehicle followed by normoxia. Ferrets were followed to P42 or P70 and underwent weekly behavioral testing consisting of negative geotaxis, cliff aversion, and righting, as well as assessment of gait coordination on an automated catwalk. At end point, hematoxylin and eosin (HE) histology, immunohistochemistry (GFAP, MBP, and Olig2), and ex vivo magnetic resonance imaging (MRI) analyses were performed.ResultsNo abnormalities were observed on HE stained sections of P11 ferrets subjected to LPS and hypoxia/hyperoxia/hypoxia, although MRI at P70 showed widespread white matter hyperintensity on T2‐weighted images, and white matter changes within the internal capsule and corpus callosum were also present on MBP and Olig2 immunohistochemistry. Behavioral testing in these animals revealed slower overall reflex development between P28 and P40, smaller hind paw areas consistent with “toe walking”, and significantly greater lateral movement and reduced gait co‐ordination. More pronounced pathology was found in animals that underwent carotid artery ligation, LPS, and hypoxia/hyperoxia/hypoxia at P17 or P21. Gross abnormalities included thinned gyri in the temporal and/or occipital lobes and widening of the longitudinal fissure, with frank areas of cystic tissue loss in the most severely affected animals. Histological abnormalities included neuronal necrosis, vacuolation and cavitation of the neuropil, and gliosis.ConclusionsFerret kits treated with LPS and hypoxia/hyperoxia/hypoxia showed pathological and motor deficits consistent with white matter injury, and more severe injury including neuronal necrosis was observed in animals subjected to bilateral carotid artery ligation. These newborn ferret models may provide an additional platform to assess potential therapies in human encephalopathy of prematurity and neonatal encephalopathy.Support or Funding InformationNIH grant 5R21NS093154‐02 (NICHD)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of acetazolamide on brain swelling and aquaporin‐4 expression in rats brain after ischemic stroke

ObjectiveTo study the effect of acetazolamide(AZA) on cerebral edema and aquaporin‐ 4 (AQP4) expression after cerebral infarction in rats. To explore the relations between ischemic cerebral edema and AQP4 expression.Methods150 male SD rats were randomly divided into three groups: sham operation group, non‐intervention group, AZA treatment group. Each group was divided into five subgroups according to the time of cerebral ischemia (6h, 1d, 3d, 5d and 7d), and there were 10 rats in each subgroup . The rats model of cerebral infarction was adopted by means of bilateral carotid arteries ligation(2‐VO) method. The treatment group was given intraperitoneal injection of AZA, while the sham operation group and the non‐intervention group were injected the same amount of normal saline at the corresponding time point above. The cerebral edema was measured by dry‐wet weight method. The expression of AQP4 in cerebral infarction was detected by immunohistochemistry.ResultsThe brain water content(BWC) and the expression of AQP4 in non‐intervention group and AZA treatment group began to increase at 6h after cerebral infarction, it reached the peak at 3d, and then decreased gradually.(P&lt;0.05). The BWC and the expression of AQP4 in sham operation group had not change significantly at any time point above (P&gt;0.05). At different time points, the BWC and the expression of AQP4 in non‐intervention group and AZA treatment group was higher than that in sham operation group.(P&lt;0.05). The BWC and the expression of AQP4 in AZA treatment group was lower than that in non‐intervention group.(P&lt;0.05). The expression of AQP4 in sham operation group had not change significantly at any time point above (P&gt;0.05).ConclusionsAcetazolamide can alleviate rats cerebral edema and the expression of AQP4 after rats cerebral infarction, and it may alleviate rats cerebral edema by reducing the expression of AQP4 in rats brain.Support or Funding InformationThis study was supported by the National Natural Science Foundation Youth Fund (30600637), China Postdoctral Foundation(2014M561207), the Shanxi Province Basic Research Program ( 2010021034‐4, 201601D011119), the Shanxi Scholarship Council of China (2011‐096 and Key Projects No.4), Department of Human Resources and Social Security of Shanxi province, The science and technology research projects of health department of Shanxi Province(2016D011119), The Teaching Reform Program of Shanxi Higher Education , The Education Reform Project of Postgraduate Students in Shanxi Province. The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Pei Yuan Tong Nao capsules on neuronal function and metabolism in cerebral ischemic rats

Ethnopharmacological significancePei Yuan Tong Nao (PYTN) capsules have been used in traditional Chinese medicine (TCM) for many centuries for the treatment of renal and vascular diseases, including cerebral ischemia. Although they are used in clinical practice, the evidence of their efficacy is lacking. Materials and methodsThe cerebral ischemic rat model was established by bilateral carotid artery ligation and shear surgery. The laser speckle blood flow imaging apparatus was used to observe the changes in cerebral blood flow before and after surgery. The working memory of rats were analyzed using the Morris water maze (MWM) test. The 2-deoxy-2-[18F]fluoro-D-glucose (18-FDG) standard uptake values (SUVs) of rats with cerebral ischemia were evaluated by small-living animal positron emission tomography (PET) imaging. The quantity of acetylcholine in the hippocampus and the protein quantity of α7 nicotinic acetylcholine receptor in the hippocampus were detected with high-resolution fluid phase chromatography and Western blot analysis. ResultsThere was no significant difference in the preoperative mean cerebral blood perfusion between the groups. There was a significant decrease (P < 0.01) in cerebral perfusion in the model-operation, nimodipine, and PYTN groups after bilateral common carotid artery occlusion (BCCAo) compared with presurgery. The escape latency is shortened in the PYTN group (P < 0.05) compared with the model-operation group. SUVs of the nimodipine and PYTN groups increased (P < 0.05) compared with the model-operation group. After treatment with nimodipine and PYTN, acetylcholine levels in the hippocampus of the rats in the respective groups were lower than that of the sham-operation group (P < 0.01), and acetylcholine levels in the hippocampus of the nimodipine and PYTN groups were higher than that of the model-operation group (P < 0.05). α7 nicotinic acetylcholine receptor analysis showed that the protein levels of the nimodipine and PYTN groups increased after treatment compared with the model-operation group (P < 0.05). ConclusionPYTN capsules improved the performance of rats with cerebral ischemia (analyzed using the MWM test), which may be due the to improvement of glucose metabolism in the hippocampus (evaluated by estimating acetylcholine and α7 nicotinic acetylcholine receptor protein).

Neuroprotective Effect of Oral Administration of Trigonella Foenum-Graecum on Chronic Cerebral Hypoperfusion in Rat Model

Introduction: Decreased cerebral blood supply to the brain can generate a condition of chronic cerebral hypoperfusion, which is one of the pathophysiological mechanisms of neuronal degeneration and cognitive impairment in Alzheimer’s disease. This study aimed to evaluate the potential neuroprotective effects of Trigonella foenum graecum seeds on chronic cerebral hypoperfusion in a rat model. Materials and Methods: Chronic cerebral hypoperfusion was induced by permanent bilateral ligation of the common carotid arteries (a two-vessel occlusion, 2VO) in male Sprague–Dawley rats. The experimental groups were divided into three groups (11 rats/group): i) sham (control) group; ii) 2VO group without any treatment; and iii) 2VO group that was administered orally with the Trigonella foenum graecum extract (100 mg/kg/day) by oral gavage from 3 days before the date of 2VO surgery and continued daily until the end of the 8th postoperative week. Spatial memory performance was assessed by the Morris water maze test. Malondialdehyde and C-reactive protein (CRP) concentration, superoxide dismutase and glutathione activities were measured in serum after 8 weeks from 2VO injury. Results: Chronic cerebral hypoperfusion rats resulted in spatial memory impairments. This behavioral dysfunction was accompanied by decreasing superoxide dismutase and glutathione activities, and increasing malondialdehyde and CRP levels in serum. Oral administration of Trigonella foenum graecum extract significantly improved the memory impairment, enhanced antioxidant enzyme activities, and decreased the malondialdehyde and CRP levels to near normal levels. Conclusion: The potential activity offered by Trigonella foenum graecum extract showed the neuroprotective effect that may be beneficial in cerebrovascular type dementia.

Evaluation of retinal injury in a rat model of transient ischemic stroke.

Stroke-associated ocular disorders are vision-threatening. This study was designed to evaluate in vivo retinal injury induced by transient global cerebral ischemia/reperfusion (I/R). A stroke-induced retinal injury model in Wistar rats was established by electrocoagulation of bilateral vertebral arteries, combined with transient ligation of the bilateral common carotid arteries. Rats were randomly divided into groups based on the time post cerebral perfusion (3 h, 24 h, 48 h, 72 h, and 7 days). Retinal injury was evaluated by histological analysis, examination of eye fundus, and TUNEL staining. The expression of protein kinase C-alpha (PKCα) and fibrillary acidic protein (GFAP) was determined using qRT-PCR and immunofluorescence analysis. Both retinal neurons and the vasculature underwent significant damage in the cerebral-I/R groups when compared to rats in the sham group. Moreover, when compared to non-stroke rats, TUNEL staining revealed signs of apoptosis in the retina after transient ischemic stroke was induced (P<0.001). In these rats, the expression of PKCα and GFAP in the retinas was enhanced and peaked at 72 h after induction of cerebral-I/R (P<0.001). In this study, we found that retinas are very susceptible to transient global cerebral-I/R injury. The expression of PKCα and GFAP may be implicated in the pathogenesis of ischemic stroke-induced retinal injury.

The expression of TREMs in ischemic optic neuropathy of rats

Objective To investigate the expression of TREMs and the related pathway in ischemic optic neuropathy (ION) of rats. Methods The permanent ligation of bilateral internal carotid arteries (BICA) of rat was performed for 14 days to establish sub-acute ION model as model group. The control group were separated BICA without ligation. The expression of TREM-1, TREM-2, Caspase-3 and IL-6 genes and proteins were detected by reverse transcription PCR(RT-PCR) and Western Blot, respectively. Results Compared with control group, the expressions of TREM-1, Caspase-3 and IL-6 in model group were higher and TREM-2 was lower (P=0.001). Conclusion TREM-1 has effects on inducing apoptosis and inflammation. Reversely, TREM-2 shows opposite effects. Key words: Optic neuropathy, ischemic; TREM-1; TREM-2; Caspase-3; IL-6

Validation of noninvasive photoacoustic measurements of sagittal sinus oxyhemoglobin saturation in hypoxic neonatal piglets.

We hypothesize that noninvasive photoacoustic imaging can accurately measure cerebral venous oxyhemoglobin saturation (So2) in a neonatal model of hypoxia-ischemia. In neonatal piglets, which have a skull thickness comparable to that of human neonates, we compared the photoacoustic measurement of sagittal sinus So2 against that measured directly by blood sampling over a wide range of conditions. Systemic hypoxia was produced by decreasing inspired oxygen stepwise (i.e., 100, 21, 19, 17, 15, 14, 13, 12, 11, and 10%) with and without unilateral or bilateral ligation of the common carotid arteries to enhance hypoxia-ischemia. Transcranial photoacoustic sensing enabled us to detect changes in sagittal sinus O2 saturation throughout the tested range of 5-80% without physiologically relevant bias. Despite lower cortical perfusion and higher oxygen extraction in groups with carotid occlusion at equivalent inspired oxygen, photoacoustic measurements successfully provided a robust linear correlation that approached the line of identity with direct blood sample measurements. Receiver-operating characteristic analysis for discriminating So2 <30% showed an area under the curve of 0.84 for the pooled group data, and 0.87, 0.91, and 0.92 for hypoxia alone, hypoxia plus unilateral occlusion, and hypoxia plus bilateral occlusion subgroups, respectively. The detection precision in this critical range was confirmed with sensitivity (87.0%), specificity (86.5%), accuracy (86.8%), positive predictive value (90.5%), and negative predictive value (81.8%) in the combined dataset. These results validate the capability of photoacoustic sensing technology to accurately monitor sagittal sinus So2 noninvasively over a wide range and support its use for early detection of neonatal hypoxia-ischemia. NEW & NOTEWORTHY We present data to validate the noninvasive photoacoustic measurement of sagittal sinus oxyhemoglobin saturation. In particular, this paper demonstrates the robustness of this methodology during a wide range of hemodynamic and physiological changes induced by the stepwise decrease of fractional inspired oxygen to produce hypoxia and by unilateral and bilateral ligation of the common carotid arteries preceding hypoxia to produce hypoxia-ischemia. This technique may be useful for diagnosing risk of neonatal hypoxic-ischemic encephalopathy.

In vivo measurement of the dynamics of norepinephrine in an olfactory bulb following ischemia-induced olfactory dysfunction and its responses to dexamethasone treatment.

Information on the dynamics of molecules following olfactory dysfunction remains essential for understanding the molecular events involved in the pathological process of olfactory dysfunction. This study for the first time demonstrates a method based on the combination of in vivo microdialysis with high performance liquid chromatography (HPLC) and electrochemical detection (ECD) for the measurement of the dynamics of norepinephrine (NE) in the olfactory bulbs of Sprague-Dawley rats following olfactory dysfunction induced by brain ischemia and its responses toward dexamethasone treatment. The method possesses a high spatial resolution and benefits from in vivo microdialysis and high selectivity and is thus capable of measuring NE in the olfactory bulb of rats. With this method, the basal level of NE in the olfactory bulb was evaluated to be ca. 235 ± 25 nM (n = 6). This level was found to increase by 260 ± 90% at a time point of 240 min after brain ischemia with bilateral ligation of both common carotid arteries. The increase was found to be suppressed upon the treatment of the animals with 0.2% dexamethasone in the olfactory bulb. These results suggest that NE is involved in the pathological process of ischemia-induced olfactory dysfunction and this information is useful to further understand the molecular events involved in olfactory dysfunction.

Signal Mechanism of the Protective Effect of Combined Preconditioning by Amtizole and Moderate Hypoxia.

The content of regulatory proteins involved in adaptation to hypoxia and ischemia was studied in brain rat homogenate under conditions of normoxia and after bilateral ligation of the common carotid arteries. Preconditioning with amtizole in combination with moderate hypoxia increased the levels of HIF-1α, erythropoietin, vascular endothelial growth factor under conditions of normoxia. During experimental ischemia, combined preconditioning led to stabilization of the content of these regulatory proteins at the level of intact control and to a decrease in glycogen synthase-3β kinase activity. This pattern of changes in regulatory proteins was noted during the early and late periods of preconditioning.

Mild hypothermia alleviates cerebral ischemia reperfusion injury via promoting neurons autophagy in mice

Objective To investigate the neurons autophagy and apoptosis after cerebral ischemia reperfusion (I/R)injury in mice, and to explore the effect of mild hypothermia on neurons autophagy and apoptosis. Methods The global cerebral ischemia-reperfusion injury model in C57 mice was established with carotid artery ligation method. Ninety-six C57 mice were randomly(random number) divided into 8 groups(n=12 in each group), namely control group (C0), sham group, normal body temperature group (NT, 37 ℃) after I/R 6 h (C6 h), normal body temperature group after I/R 12 h (C12 h), normal body temperature group after I/R 72 h(C72 h), mild hypothermia group (MH, 34 ℃) after I/R6 h (C6 h+ MH), mild hypothermia group after I/R12 h (C12 h+ MH), mild hypothermia group after I/R 72 h (C72 h+ MH). The protein expressions of Sirt1, P-FoxO1, Rab7, P53 and autophagy related genes such as Beclin1, LC3 were detected by Western blot at given intervals. The LC3 granules were assayed by immunofluorescence. The neurons apoptosis was detected by TUNEL. The software of SPSS13.0 was used for statistical analysis. Measurement data was expressed with mean±SD, and comparison between two groups was carried out with Student’s t test, One-way ANOVA was used for comparisons among groups, and P<0.05 was considered statistically significant. Results The global cerebral ischemia-reperfusion injury model in C57 mice was established successfully with bilateral carotid arteries ligation method. Compared with the control group, the protein expressions of Sirt1, P-FoxO1, Rab7, Beclin1 and LC3Ⅱ/Ⅰwere gradually reduced, especially at 12 h after I/R in NT group (P<0.05), while the expression of P53 was obviously increased (P<0.05). In MH group, the expressions of Sirt1, P-FoxO1, Rab7, Beclin1, LC3Ⅱ/Ⅰwere higher than those in NT group (P< 0.05). And the expression of P53 was lower than that in NT group (P<0.05). Immuno-fluorescence test showed that compared with the control group, the LC3 particles of neurons cells were decreased significantly in group C12(at 12 h after I/R 6.0±1.5 vs. 18.1±2.5, P<0.05). Nevertheless, LC3 particles were increased in MH group compared with NT group(36.1±4.5 vs. 6.0±1.5, P<0.05). The results of TUNEL test showed that compared with the control group, neurons cells apoptosis were significantly increased in C72 group(at 72 h after I/R, 54.8%±7.5% vs. 5.5%±1.2%, P<0.05). However, compared with NT group, neurons apoptosis were decreased in MH group(28.8%±4.5% vs. 54.8%±7.5%, P<0.05). Conclusions The neuron autophagy was significantly reduced and apoptosis was significantly increased after ischemia reperfusion injury (I/R) in mice. However, mild hypothermia could increase the expression of Sirt1, FoxO1, beclin1 and LC3, so as to promote neurons autophagy and reduce apoptosis, which would provide therapy target for neurons injury after hypoxia and provide soundly theoretical basis for mild hypothermia for clinical application. Key words: Ischemia reperfusion; Mild hypothermia; Neurons; Autophagy; Apoptosis

Dynamic Changes in the Renin-Angiotensin-Aldosterone System and the Beneficial Effects of Renin-Angiotensin-Aldosterone Inhibitors on Spatial Learning and Memory in a Rat Model of Chronic Cerebral Ischemia.

Renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation of blood pressure and brain function. Therefore, we studied the dynamic changes in the RAAS in the blood, cerebral cortex, and hippocampus and the effects of RAAS inhibitors on spatial learning and memory and hippocampal apoptosis in a rat model of chronic cerebral ischemia (CCI) established by bilateral ligation of the common carotid arteries of rats. The levels of renin, angiotensin II (Ang II), and aldosterone (ALD) in the plasma, and the homogenates of the left side of cerebral cortex and whole hippocampus of rats were detected on day 1, 3, 7, 14, 21, and 30 by radioimmunoassay. Spatial learning and memory and hippocampal apoptosis were evaluated on day 30 by Morris water maze test (navigation and space exploration tests) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, respectively, after rats were orally administered with distilled water (DW), renin inhibitor aliskiren (30 mg/kg), Ang converting enzyme inhibitor enalapril (4 mg/kg), or Ang II receptor antagonist candesartan (2 mg/kg) daily for 30 days. The results showed that the levels of renin and Ang II were significantly higher but ALD fluctuated in the blood, cerebral cortex, and hippocampus in CCI rats compared to normal rats. However, aliskiren and enalapril could significantly decrease (p < 0.05) the levels of renin, Ang II and ALD in the blood, cerebral cortex, and hippocampus compared to DW treatment; while candesartan had similar effect on renin and ALD but no effect on Ang II in CCI rats. Furthermore, spatial learning and memory were significantly decreased but apoptosis in the hippocampus was obviously increased in CCI rats compared to normal rats (p < 0.05). However, aliskiren, enalapril, and candesartan were equally effective to improve spatial learning and memory and decrease apoptosis in the hippocampus. Therefore, RAAS plays an important role in the development of cerebral ischemia and RAAS inhibitors aliskiren, enalapril, and candesartan improve spatial learning and memory and protect brain injury by inhibiting hippocampal apoptosis in CCI rats.