Unilateral Common Carotid Artery Ligation Research Articles

Urotensin II receptor deficiency ameliorates ligation-induced carotid intimal hyperplasia partially through the RhoA-YAP1 pathway

Intimal hyperplasia (IH) is a common pathological feature of vascular proliferative diseases, such as atherosclerosis and restenosis after angioplasty. Urotensin II (UII) and its receptor (UTR) are widely expressed in cardiovascular tissues. However, it remains unclear whether the UII/UTR system is involved in IH. Right unilateral common carotid artery ligation was performed and maintained for 21 days to induce IH in UTR knockout (UTR−/−) and wild-type (WT) mice. Histological analysis revealed that compared with WT mice, UTR-deficient mice exhibited a decreased neointimal area, angiostenosis and intima–media ratio. Immunostaining revealed fewer smooth muscle cells (SMCs), endothelial cells and macrophages in the lesions of UTR−/− mice than in those of WT mice. Protein interaction analysis suggested that the UTR may affect cell proliferation by regulating YAP and its downstream target genes. In vitro experiments revealed that UII can promote the proliferation and migration of SMCs, and western blotting also revealed that UII increased the protein expression of RhoA, CTGF, Cyclin D1 and PCNA and downregulated p-YAP protein expression, while these effects could be partly reversed by urantide. To evaluate the translational value of UTRs in IH management, WT mice were also treated with two doses of urantide, a UTR antagonist, to confirm the benefit of UTR blockade in IH progression. A high dose of urantide (600 μg/kg/day), rather than a low dose (60 μg/kg/day), successfully improved ligation-induced IH compared with that in mice receiving vehicle. The results of the present study suggested that the UII/UTR system may regulate IH partly through the RhoA-YAP signaling pathway.

Development of a new cerebral ischemia reperfusion model of Mongolian gerbils and standardized evaluation system.

The Mongolian gerbil is an excellent laboratory animal for preparing the cerebral ischemia model due to its inherent deficiency in the circle of Willis. However, the low incidence and unpredictability of symptoms are caused by numerous complex variant types of the circle. Additionally, the lack of an evaluation system for the cerebral ischemia/reperfusion (I/R) model of gerbils has shackled the application of this model. We created a symptom-oriented principle and detailed neurobehavioral scoring criteria. At different time points of reperfusion, we analyzed the alteration in locomotion by rotarod test and grip force score, infarct volume by triphenyltetrazolium chloride (TTC) staining, neuron loss using Nissl staining, and histological characteristics using hematoxylin-eosin (H&E) straining. With a successful model rate of 56%, 32 of the 57 gerbils operated by our method harbored typical features of cerebral I/R injury, and the mortality rate in the male gerbils was significantly higher than that in the female gerbils. The successfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion; formed obvious infarction; exhibited typical pathological features, such as tissue edema, neuronal atrophy and death, and vacuolated structures; and were partially recovered with the extension of reperfusion time. This study developed a new method for the unilateral common carotid artery ligation I/R model of gerbil and established a standardized evaluation system for this model, which could provide a new cerebral I/R model of gerbils with more practical applications.

In vivo monitoring of hemodynamic changes in ischemic stroke using photoacoustic tomography.

Ischemic stroke occurs when a blood vessel supplying the brain is blocked, leading to decreased blood flow. Early diagnosis and treatment are crucial. However, existing clinical imaging methods have limitations, such as safety issues and low time resolution. To address these challenges, we propose using photoacoustic tomography (PAT) with a contrast agent, known for its high resolution and contrast capabilities. Our study involved imaging brain vasculature in three groups: normal, unilateral common carotid artery ligation (UCAL), and middle cerebral artery occlusion (MCAO). On the ischemic stroke side, we observed reduced blood vessel density and hemodynamic changes were evident after injecting indocyanine green for PAT. The photoacoustic intensity was notably lower in the ligated sides of the UCAL and MCAO groups, with statistically significant differences between the three groups. This work highlights PAT's potential as a powerful tool for early diagnosis and guidance in ischemic stroke cases.

Periventricular Microglia Polarization and Morphological Changes Accompany NLRP3 Inflammasome-Mediated Neuroinflammation after Hypoxic-Ischemic White Matter Damage in Premature Rats.

White matter damage (WMD) is a primary cause of cerebral palsy and cognitive impairment in preterm infants, and no effective treatments are available. Microglia are a major component of the innate immune system. When activated, they form typical pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes and regulate myelin development and synapse formation. Therefore, they may play a pivotal role in hypoxic-ischemic (HI) WMD. Herein, we investigated neural inflammation and long-term microglia phenotypic polarization in a neonatal rat model of hypoxia-ischemia-induced WMD and elucidated the underlying pathophysiological processes. We exposed 3-day-old (P3) Sprague-Dawley rats to hypoxia (8% oxygen) for 2.5 hr after unilateral common carotid artery ligation. The activation of NLRP3 inflammatory bodies, microglia M1/M2 polarization, myelination, and synaptic development in our model were monitored 7, 14, and 21 days after birth. In addition, the Morris water maze test was performed on postnatal Day 28. We confirmed myelination disturbance in the periventricular white matter, abnormal synaptic development, and behavioral changes in the periventricular area during the development of HI WMD. In addition, we found an association between the occurrence and development of HI WMD and activation of the NLRP3 inflammasome, microglial M1/M2 polarization, and the release of inflammatory factors. NLRP3 inhibition can play an anti-inflammatory role by inhibiting the differentiation of microglia into the M1 phenotype, thereby improving myelination and synapse formation. In conclusion, microglia are key mediators of the inflammatory response and exhibit continuous phenotypic polarization 7-21 days after HI-induced WMD. This finding can potentially lead to a new treatment regimen targeting the phenotypic polarization of microglia early after HI-induced brain injury.

Flavonoids from Citrus paradise cv. Changshan-huyou exerts protective effect on ischemia-induced cerebral injury in mice via inhibiting RhoA-ROCK2 signaling pathway.

To investigate the protective effect and mechanism of total flavonoids from Citrus paradise cv. Changshan-huyou extracts (TFC) on oxygen-glucose deprivation (OGD) of primary neurons and chronic ischemia-induced cerebral injury in mice. Primary hippocampal neurons of 18-day fetal rats were isolated and cultured for 1 week, then treated with 0.25, 0.50 and 1.00 mg/mL TFC. After oxygen-glucose deprivation for 1 h, cells were reperfused for 6 and 24 h, respectively. The cytoskeleton was observed by phalloidin staining. In animal study, 6-week ICR male mice were randomly divided into sham operation group, model group, low-dose (10 mg/kg), medium-dose (25 mg/kg) and high-dose (50 mg/kg) TFC treatment groups, with 20 mice in each group. After 3 weeks, chronic cerebral ischemia was induced by unilateral common carotid artery ligation in all groups except sham operation group. Mice were treated with different concentrations of TFC in the three TFC treatment groups for 4 weeks. Open field test, novel object recognition test and Morris water maze test were used to evaluate anxiety, learning and memory of these mice. Nissl, HE and Golgi stainings were used to detect neuronal degeneration and dendritic spine changes in the cortex and the hippocampus. The expression levels of Rho-associated kinase (ROCK) 2, LIM kinase (LIMK) 1, cofilin and its phosphorylation, as well as the expression of globular actin (G-actin) and filamentous actin (F-actin) protein in hippocampus of mice were detected by Western blotting. Neurons subjected to OGD showed that neurites displayed shortening and breakage; while treatment with TFC reversed OGD-induced neurite injury, especially in the 0.50 mg/mL TFC group. Compared with the sham operation group, the mice in the model group showed a significant decline in anxiety and cognitive ability (P<0.01), whereas treatment with TFC significantly reversed anxiety and cognitive deficits (P<0.05). Improvement in the medium-dose TFC group was the most obvious. Histopathological analysis indicated that the number of Nissl bodies and dendritic spines in hippocampus and cortex were decreased in the model group (all P<0.01). However, after treatment with medium dose of TFC, the number of Nissl bodies and dendritic spines (all P<0.05) was significantly recovered. Compared with the sham operation group, the phosphorylation level of ROCK2 in the brain tissue of the model group was significantly increased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly decreased (P<0.05), and the relative content ratio of G-actin/F-actin was significantly increased (P<0.05). After administration of TFC, the phosphorylation level of ROCK2 in brain tissue of each group was significantly decreased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly up-regulated (P<0.05) and the relative content ratio of G-actin/F-actin was significantly decreased (P<0.05). TFC protects from ischemia-induced cytoskeletal damage, reduces neuronal dendritic spine injury and protects mice against chronic cerebral ischemia through RhoA-ROCK2 signaling pathway, indicating that TFC might be a potential candidate for treatment of chronic ischemic cerebral injury.

Improving accuracy of cerebral blood flow measurements in laser speckle flowmetry

Laser speckle flowmetry (LSF) can measure moving red blood cells in real time as changes in tissue blood flow; however, LSF values may not be sufficiently reproducible due to body movements and other factors. Therefore, it is difficult to compare absolute values of LSF directly. This study aimed to verify the influence of head fixation on improving the measurement accuracy and enabling the comparison of LSF values semi-quantitatively using an external standard. We first examined the effects of head fixation on LSF values obtained during cerebral blood flow (CBF) measurement through a bone window over the cerebrum in Sprague-Dawley rats. The LSF values in the bone window were semi-quantitated using the ratio of the external standard LSF values measured simultaneously in the same field of view in a unilateral common carotid artery ligation model to evaluate CBF. Head fixation halved the variability of LSF values when compared with measurements made without head fixation. The LSF laser focus position did not affect the LSF values provided the focus was positioned within the exposed cranium. Semi-quantitation of LSF values made it possible to monitor changes in CBF in each respective bone window over time in a model of unilateral carotid ligation. In conclusion, the stabilizing effect of head fixation on LSF values was clarified, and we observed that to improve measurement accuracy, the head should be immobilized. Semi-quantitation of LSF values was first introduced by using an external standard and appears to be useful in monitoring the CBF of each location separately.

Glucocorticoid Receptor Blockers Pretreatment Did Not Improve Infarct Volume in Type-2 Diabetic Mouse Model of Stroke

Impaired glucocorticoid signaling in diabetes mellitus and its relation to suppressed immune function and hyperglycemia during acute stroke has been shown to be detrimental. Therefore, the aim of this study was to examine the effect of glucocorticoid receptor (GCR) blockers in a type-2 diabetic mouse model following hypoxia–ischemia (HI). We induced stroke in diabetic db/db and non-diabetic db/+ mice by unilateral common carotid artery ligation followed by 20 min of HI. Mice were pretreated with RU-486, GCRII blocker (40 mg/kg), intraperitoneally, the day before, during stroke and post-HI. Blood and brain samples were collected at 24 h post-HI to measure blood glucose, corticosterone and infarct size. Similarly, another set of mice was pretreated with RU-486 + spironolactone, GCR1 blocker (25 mg/kg) subcutaneously for a week before inducing stroke and during recovery. Samples were collected at 48 h post-HI for various analyses. RU-486 treatment did not lower the blood glucose significantly, but RU-486 + spironolactone decreased the blood glucose in db/db mice post-HI. However, none of the treatment groups decreased the ischemia-induced serum corticosterone level or infarct size. This study suggests that even though GCR blockers improve hyperglycemia, they did not improve the infarct volume.

Key Phytochemicals and Biological Functions of Chuanxiong Rhizoma Against Ischemic Stroke: A Network Pharmacology and Experimental Assessment.

Presently, the treatment options for ischemic stroke (IS) are limited due to the complicated pathological process of the disease. Chuanxiong Rhizome (CR), also known as Conioselinum anthriscoides “Chuanxiong” (rhizome), is the most widely used traditional Chinese medicine for treating stroke. This study aimed to uncover the key phytochemicals and biological functions of CR against IS through a network pharmacology approach combining with IS pathophysiology analysis. We employed permanent unilateral common carotid artery ligation to construct a mouse model of global cerebral ischemia and found that cerebral ischemia injuries were improved after 7 days of gavage treatment of CR (1,300 mg/kg/day). CR exerts protective effects on neurons mainly by acting on targets related to synaptic structure, synaptic function, neuronal survival and neuronal growth. A total of 18 phytochemicals from CR based on UHPLC-MS/MS that corresponded to 85 anti-IS targets. Coniferyl ferulate, neocnidilide and ferulic acid were identified as the key phytochemicals of CR against IS. Its brain protective effects involve anti-inflammatory, anti-oxidative stress, and anti-cell death activities and improves blood circulation. Additionally, the two most important synergistic effects of CR phytochemicals in treating IS are prevention of infection and regulation of blood pressure. In brain samples of Sham mice, L-tryptophan and vanillin were detected, while L-tryptophan, gallic acid, vanillin and cryptochlorogenic acid were detected in IS mice by UHPLC–MS/MS. Our findings provide a pathophysiology relevant pharmacological basis for further researches on IS therapeutic drugs.

Deleterious Effect of Crossfostering in Rat Pups on Hypoxic-Ischaemic Injury Tolerance and Hypothermic Neuroprotection

We study the effect of hypothermia (HT) following hypoxic-ischaemic (HI) brain injury in postnatal day 7 (P7) rats. In 2015, new European Union animal transport regulations prompted a change in practice at the breeding facility, which henceforth crossfostered P3 litters to P8 older lactating dams prior to transportation. It is generally assumed that crossfostering does not significantly affect the experimental results. The aim of this study was to examine whether crossfostering affects our model consistency by modifying injury susceptibility and hypothermic neuroprotection. We analysed 219 pups from 11 experiments conducted between 2013 and 2015: 73 non-crossfostered and 146 crossfostered pups. At P7, all pups underwent unilateral common carotid artery ligation followed by 50 min of hypoxia (8% O₂, 36°C). Immediately after this mild insult, the pups were randomized to post-insult normothermia or HT treatment. Pups were culled at P14. Injury was assessed by area loss of the ipsilateral hemisphere and histopathology scoring of the hippocampus, cortex, thalamus, and basal ganglia. Crossfostered pups had double the injury compared to non-crossfostered pups irrespective of the treatment group. Hypothermic neuroprotection was statistically significant, but with a smaller and less consistent effect in crossfostered pups (relative neuroprotection 16% vs. 31% in non-crossfostered). These results demonstrate hypothermic neuroprotection following a mild HI insult. A representative subset of 41 animals was also assessed for evidence of microglial reactivity; however, no detectable difference in microglial reactivity was observed between any of the groups. In conclusion, crossfostering alters outcomes in our established model through reduced insult tolerance and variable neuroprotection. Crossfostering as a common breeding practice is a largely unexplored variable in animal research that may result in invalid research conclusions if inadequately adjusted for by larger group sizes. As a result, crossfostering is likely to be inconsistent with the principles of replacement, reduction, and refinement.

Pyk2 inhibition attenuates hypoxic-ischemic brain injury in neonatal mice.

Newborns suffering from hypoxia-ischemia (HI) brain injury still lack effective treatment. Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor tyrosine kinase, which is highly correlated with transient ischemic brain injury in adult. In this study, we investigated the role of Pyk2 in neonatal HI brain injury. HI was induced in postnatal day 7 mouse pups by unilateral common carotid artery ligation followed by hypoxic exposure. Pyk2 interference lentivirus (LV-Pyk2 shRNA) was constructed and injected into unilateral cerebral ventricle of neonatal mice before HI. Infarct volume, pathological changes, and neurological behaviors were assessed on postnatal day 8-14. We showed that the phosphorylation level of Pyk2 was significantly increased in neonatal brain after HI, whereas LV-Pyk2 shRNA injection significantly attenuated acute HI brain damage and improved neurobehavioral outcomes. In oxygen-glucose deprivation-treated cultured cortical neurons, Pyk2 inhibition significantly alleviated NMDA receptor-mediated excitotoxicity; similar results were also observed in neonatal HI brain injury. We demonstrated that Pyk2 inhibition contributes to the long-term cerebrovascular recovery assessed by laser speckle contrast imaging, but cognitive function was not obviously improved as evaluated in Morris water maze and novel object recognition tests. Thus, we constructed lentiviral LV-HIF-Pyk2 shRNA, through which HIF-1α promoter-mediated interference of Pyk2 would occur during the anoxic environment. Intracerebroventricular injection of LV-HIF-Pyk2 shRNA significantly improved long-term recovery of cognitive function in HI-treated neonatal mice. In conclusion, this study demonstrates that Pyk2 interference protects neonatal brain from hypoxic-ischemic injury. HIF-1α promoter-mediated hypoxia conditional control is a useful tool to distinguish between hypoxic period and normal period. Pyk2 is a promising drug target for potential treatment of neonatal HI brain injury.

History and Recent Development of Basic Research Using an Experimental Intracranial Aneurysm Model

Since intracranial aneurysm(IA)is a disease that follows an extremely unpredictable course, from initiation to rupture, experimental models have greatly contributed to a better understanding of IA pathophysiology. This article aims to review the history of IA models through the pivotal theme of the ideal IA model. In addition, this article introduces updated findings from the application of these experimental models. Though the first IA model, known as a venous pouch model, was reported in 1954, it mimicked only the shape of the IA, without reproducing its pathological structure or blood-flow characteristics. Currently, two models are generally applied: the "Hashimoto model," produced by unilateral common carotid artery(CCA)ligation followed by systemic hypertension and weakening of the vascular wall, and the "elastase injection model," induced by intraventricular elastase injection and also followed by systemic hypertension. In addition, other models, including a rabbit basilar top IA, developed after bilateral CCA ligation, and an artificial bifurcation model, generated by an anastomosis between the CCAs, have been found to be valuable for computational fluid dynamics analysis. Through this advancement, the IA model has gradually elucidated the pathophysiology of IA as a flow-induced inflammatory disease. Nowadays, vascular inflammation is suggested to be regulated by bacterial flora. Further development of IA models and a better understanding of IA pathophysiology are expected in the future.

Artemin Is Upregulated by TrkB Agonist and Protects the Immature Retina Against Hypoxic-Ischemic Injury by Suppressing Neuroinflammation and Astrogliosis.

Hypoxic-ischemia (HI) is a major cause of acquired visual impairment in children from developed countries. Previous studies have shown that systemic administration of 7,8-dihydroxyavone (DHF), a selective tropomyosin receptor kinase B (TrkB) agonist, provides long-term neuroprotection against HI injury in an immature retina. However, the target genes and the mechanisms of the neuroprotective effects of TrkB signaling are not known. In the present study, we induced an HI retinal injury through unilateral common carotid artery ligation followed by 8% oxygen for 2 h in P7 rat pups. DHF was administered intraperitoneally 2 h before and 18 h after the HI injury. A polymerase chain reaction (PCR) array was used to identify the target genes upregulated after the DHF treatment, which was then confirmed with quantitative real-time reverse transcriptase PCR and a western blot. Effects of the downstream mediator of DHF were assessed using an intravitreal injection of neutralizing antibody 4 h after DHF administration (24 h after HI). Meanwhile, the target protein was injected into the vitreous 24 h after HI to validate its protective effect when exogenously supplemented. We found that systemic DHF treatment after HI significantly increased the expression of the artemin (ARTN) gene and protein at P8 and P10, respectively. The neuroprotective effects of DHF were inhibited after the ARTN protein blockade, with an increase in neuroinflammation and astrogliosis. ARTN treatment showed long-term protection against HI injury at both the histopathological and functional levels. The neuroprotective effects of ARTN were related to a decrease in microglial activation at P17 and attenuation of astrogliosis at P29. ARTN enhances phosphorylation of RET, ERK, and JNK, but not AKT or p38 in the immature retina. Altogether, these results suggest that the neuroprotective effect of a TrkB agonist is partially exerted through a mechanism that involves ARTN because the protective effect is ameliorated by ARTN sequestration. ARTN treatment after HI injury protects the immature retina by attenuating late neuroinflammation and astrogliosis in the immature retina relating to the ARTN/RET/JNK/ERK signaling pathway. ARTN may be a strategy by which to provide long-term protection in the immature retina against HI injury.

Brain damage caused by neonatal hypoxia-ischemia and the effects of hypothermia in severe combined immunodeficient (SCID) mice

Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of brain damage in newborns. Although therapeutic hypothermia has been shown to be neuroprotective against neonatal HIE in clinical trials, its effect is not satisfactory. Cell-based therapies have attracted much attention as novel treatments for HIE. Preclinical studies on a variety of human cell transplantation methods have been performed in immunodeficient/immunosuppressed animals, such as severe combined immunodeficient (SCID) mice, which lack functional T and B lymphocytes. The detailed characteristics of neonatal HIE in SCID mice, however, have not been delineated. In preclinical studies, novel therapies for neonatal HIE should be evaluated in combination with hypothermia, which has become a standard treatment for neonatal HIE. However, the effects of hypothermia in SCID mice have not been delineated. In the present study, we compared neonatal hypoxic-ischemic (HI) brain damage in SCID mice and wild-type mice treated with or without hypothermia. Male and female mouse pups were subjected to HI insult induced by unilateral common carotid artery ligation combined with systemic hypoxia on postnatal day 12. In the first 4 h after HI insult, body temperature was maintained at 36 °C for the normothermia groups or 32 °C for the hypothermia groups. The severity of brain damage in SCID mice did not differ from that in wild-type mice based on most evaluations, i.e., cerebral blood flow, hemiparesis, muscle strength, spontaneous activity, cerebral hemispheric volume, neuropathological injury, and serum cytokine levels, although spleen weight, brain weight, leukocyte counts and the levels of some cytokines in the peripheral blood were different between genotypes. The effects of hypothermia in SCID mice were comparable to those in wild-type mice based on most evaluations. Taken together, these findings indicate that SCID mice can be used as an appropriate preclinical model for cell therapies for neonatal HIE.

Neuroprotective role of Nrf2 on hypoxic-ischemic brain injury in neonatal mice.

Inflammation and oxidative stress play a key role in mediating the pathophysiology of hypoxic-ischemic (HI) brain injury. Nrf2 is a transcriptional factor that contributes to the innate defense of the body against oxidative stress and inflammation. The current study investigated the effect of Nrf2 in neonatal HI brain injury using Nrf2-/- mice. Nrf2-/- and wild-type Nrf2+/+ mice on a C57BL/6J background at postnatal day 9 underwent unilateral common carotid artery ligation, followed by hypoxia. Brain damage was determined by infarct size measurement. Apoptosis was evaluated by measuring the expression of Bax and Bcl-2. The levels of inflammatory cytokines and mediators involved in oxidative stress were measured. Nrf2 knockout exacerbated HI injury-induced brain infarct and cell apoptosis in the brain. Nrf2-/- mice showed increased inflammatory cytokines and MDA, and reduced activities of antioxidant enzymes including CAT, GSH-Px, and SOD. Nrf2-/- mice showed reduced HO-1 expression after HI injury compared with wild-type mice. This study supported a protective effect of Nrf2 in neonatal HI brain injury.

Neuromodulatory Effect of NLRP3 and ASC in Neonatal Hypoxic Ischemic Encephalopathy

Background: Following birth asphyxia there is a robust inflammatory response. NLRP3 is a receptor of the innate immune system. Upon activation, NLRP3 forms an inflammasome together with ASC and procaspase-1 to mediate release of IL-1β and IL-18. NLRP3 has previously been shown to be upregulated following neonatal hypoxic-ischemic (HI) brain injury in mice, but with no early effect on brain injury. Objective: We aimed to evaluate if deficiency of NLRP3 or ASC protects against neonatal HI brain damage 7 days after hypoxia-ischemia. Methods: C57BL/6J, NLRP3^–/–, and ASC^–/– mice were subjected to unilateral common carotid artery ligation followed by hypoxia at P9. Brain infarction, apoptosis, and microglial response were evaluated, as well as total RNA sequencing and examination of plasma levels of systemic proinflammatory cytokines. Results: NLRP3^–/– mice showed significantly increased brain infarction volumes compared to wild-type (Wt) mice, while ASC^–/– mice showed reduced brain infarction volumes after neonatal hypoxia-ischemia. The amount of activated microglia was increased in NLRP3^–/– mice, while decreased in ASC^–/– mice compared to Wt mice. Total RNA sequencing showed an impaired inflammatory transcriptional response in the hippocampus of NLRP3^–/– mice. Plasma levels of IL-1β and IL-18 were not affected, but TNF was lower in NLRP3^–/– and ASC^–/– mice compared to Wt mice. Conclusion: ASC deficiency is neuroprotective in neonatal HI brain damage in mice, while NLRP3 deficiency increases brain damage.

Short-, Mid-, and Long-Term Effect of Granulocyte Colony-Stimulating Factor/Stem Cell Factor and Fms-Related Tyrosine Kinase 3 Ligand Evaluated in an In Vivo Model of Hypoxic-Hyperoxic Ischemic Neonatal Brain Injury.

Hematopoietic growth factors are considered to bear neuroprotective potential. We have previously shown that delayed treatment with granulocyte colony-stimulating factor (G-CSF)/stem cell factor (SCF) and Fms-related tyrosine kinase 3 ligand (FL) ameliorates excitotoxic neonatal brain injury. The effect of these substances in combined-stressor neonatal brain injury models more closely mimicking clinical conditions has not been investigated. The aim of this study was to assess the short-, mid-, and long-term neuroprotective potential of G-CSF/SCF and FL in a neonatal model of hypoxic-hyperoxic ischemic brain injury. Five-day-old (P5) CD-1 mice were subjected to unilateral common carotid artery ligation and subsequent alternating periods of hypoxia and hyperoxia for 65 minutes. Sixty hours after injury, pups were randomly assigned to intraperitoneal treatment with (i) G-CSF (200 μg/kg)/SCF (50 μg/kg), (ii) FL (100 μg/kg), or (iii) vehicle every 24 hours for three or five consecutive days. Histopathological and functional outcomes were evaluated on P10, P18, and P90. Baseline outcome parameters were established in sham-treated and healthy control animals. Gross brain injury did not significantly differ between treatment groups at any time point. On P10, caspase-3 activation and caspase-independent apoptosis were similar between treatment groups; cell proliferation and the number of BrdU-positive vessels did not differ on P18 or P90. Neurobehavioral assessment did not reveal significant differences between treatment groups in accelerod performance, open field behavior, or novel object recognition capacity on P90. Turning behavior was more frequently observed in G-CSF/SCF- and FL-treated animals. No sex-specific differences were detected in any outcome parameter evaluated. In hypoxic-hyperoxic ischemic neonatal brain injury, G-CSF/SCF and FL treatment does not convey neuroprotection. Prior to potential clinical use, meticulous assessment of these hematopoietic growth factors is mandated.

Clinical characteristics of internal carotid artery pseudoaneurysms in the sphenoid sinus

BackgroundInternal carotid artery (ICA) pseudoaneurysms are associated with high mortality if lack of proper management. Patients with ICA pseudoaneurysms in the sphenoid sinus often visit a hospital's ear, nose and throat (ENT) department due to nasal bleeding. In such cases, simple examination and therapy will lead to misdiagnosis. ObjectiveThis study sought to investigate the clinical characteristics, diagnostic methods and treatment of ICA pseudoaneurysms in the sphenoid sinus. MethodsVarious data, including clinical features, imaging examination results, and treatment and prognosis information, were collected and analyzed for 8 patients who visited the Department of Otolaryngology, Head & Neck Surgery of West China Hospital from March 2008 to January 2017. ResultsThe patients included 6 males and 2 females (ages 16 to 56 years). Repeated epistaxis was a common symptom in six of the eight patients (6/8), whereas monocular blindness and binocular blindness were observed in the other two patients. Head trauma was found to play a role in the induction of ICA pseudoaneurysms, given that five patients (5/8) exhibited a specific history of head injury. CT examination tended to result in misdiagnosis, whereas MRI and digital subtraction angiography (DSA) were helpful for obtaining a definite diagnosis with all diagnoses were confirmed via DSA. Coated stent intervention was performed in five patients, while carotid artery ligation was performed in two patients in emergency situations: one of whom exhibited paraplegia, but recovery was ultimately observed after rehabilitation. Moreover, failure of coated stent intervention in one patient was resolved via additional unilateral common carotid artery ligation. Furthermore, one patient with vision loss experienced vision restoration. One patient discontinued treatment for personal reasons and was lost to follow-up. No recurrence was observed in the other 7 patients. ConclusionICA pseudoaneurysms in the sphenoid sinus are uncommon. To accurately identify ICA pseudoaneurysms, collaboration between otolaryngologists and imaging specialists is essential. On the other hand, both surgical and interventional treatments can achieve good results; therefore, otolaryngologists should enhance their cooperation with neurosurgery and intervention departments. Accurate diagnosis and rapid treatment are keys to managing ICA pseudoaneurysms.

Abstract 714: Does the Sickle Trait Portend Increased Cardiovascular Risks?

Objectives: Despite the increased incidence and worse outcomes of cardiovascular diseases among African-Americans compared to the general population, the contribution of sickle cell trait (AS) to this racial disparity remains unknown. We aimed to delineate the impact of AS on the pathogenesis of cardiovascular diseases alone, and in the setting of other risk factors. Methods: We compared multiple cardiovascular endpoints between AS and wild-type genotype (AA) using the humanized Townes sickle cell mouse model. To determine whether AS modifies a pre-existing cardiovascular risk factor, diabetes was induced in a cohort of mice using streptozotocin (STZ). Hind limb ischemia model was employed to evaluate collateral vessel formation, and perfusion recovery was characterized with LASER Doppler perfusion imaging (LDPI). The running wheel assay assessed spontaneous motor function after ischemia. Angiotensin II (Ang II) and L-NAME were used to evaluate hypertensive response. Experimental stroke was induced by unilateral common carotid artery ligation and daily 15 min exposure to 7.5% oxygen for 3 days, and the infarct size was quantified as the ratio of the infarcted area to the total area of the uninjured contralateral hemisphere. PCSK9 gain-of-function mutation in the setting of Ang II and high-fat diet was used to evaluate atherosclerosis development. Results: 4 groups of mice were studied: AA, AS, AA-STZ and AS-STZ. The formation of collateral vessels was equivalent between the AA and AS (day 28, AA: 80 ± 6%, AS: 85 ± 5%, p=0.8), and in the setting of diabetes (39 ± 5% AA-STZ; 36 ± 4% AS-STZ). Systolic blood pressure at baseline (110 ± 4 mmHg vs 112 ± 5 mmHg), and in response to Ang II (148 ± 4 mmHg vs 142 ± 5 mmHg) and L-NAME (135 ± 3mmHg vs 138 ± 5 mmHg) were equivalent. Mortality from stroke was equivalent between AA and AS. After 1 month of PCKS9 gain-of-function mutation/high-fat diet, AA and AS demonstrated similar lipid profiles. Mortality and infarct volume after stroke induction were identical between AA and AS. Conclusions: All major indices of cardiovascular outcomes were similar between AA and AS, even in the presence of diabetes. These various experimental models suggest that the sickle trait likely does not contribute significantly to the poor cardiovascular outcomes seen in the African-American population.

Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

Hypoxic-ischemic injury (HI) to the neonatal human brain results in myelin loss that, in some children, can manifest as cerebral palsy. Previously, we had found that neuronal overexpression of the bone morphogenic protein (BMP) inhibitor noggin during development increased oligodendroglia and improved motor function in an experimental model of HI utilizing unilateral common carotid artery ligation followed by hypoxia. As BMPs are known to negatively regulate oligodendroglial fate specification of neural stem cells and alter differentiation of committed oligodendroglia, BMP signaling is likely an important mechanism leading to myelin loss. Here, we showed that BMP signaling is upregulated within oligodendroglia of the neonatal brain. We tested the hypothesis that inhibition of BMP signaling specifically within neural progenitor cells (NPCs) is sufficient to protect oligodendroglia. We conditionally deleted the BMP receptor 2 subtype (BMPR2) in NG2-expressing cells after HI. We found that BMPR2 deletion globally protects the brain as assessed by MRI and protects motor function as assessed by digital gait analysis, and that conditional deletion of BMPR2 maintains oligodendrocyte marker expression by immunofluorescence and Western blot and prevents loss of oligodendroglia. Finally, BMPR2 deletion after HI results in an increase in noncompacted myelin. Thus, our data indicate that inhibition of BMP signaling specifically in NPCs may be a tractable strategy to protect the newborn brain from HI.

Administration of secretoneurin is protective in hypoxic–ischemic neonatal brain injury predominantly in the hypoxic-only hemisphere

Neonatal brain injury is a problem of global importance. To date, no causal therapies are available. A substance with considerable therapeutic potential is the endogenous neuropeptide secretoneurin (SN), which has proven to be beneficial in adult stroke. The aim of this study was to assess its effect in neonatal hypoxic–ischemic brain injury models. In vitro, primary hippocampal neurons were pre-treated with vehicle, 1µg/ml, 10µg/ml, or 50µg/ml SN and subjected to oxygen–glucose deprivation (OGD) for six hours. Cell death was assessed after a 24-h recovery period. In vivo, seven day-old CD-1 mice underwent unilateral common carotid artery ligation and were exposed to 8% oxygen/nitrogen for 20 min. SN plasma concentrations were serially determined by ELISA after insult. One hour after hypoxia, a subgroup of animals was treated with vehicle or SN. SN plasma concentrations significantly decreased 48h after insult. The number of caspase-3-positive cells was significantly lower in the hypoxic–ischemic hemisphere in the thalamus of SN-treated animals. In the hypoxic-only hemisphere administration of SN significantly reduced the number of caspase-3-positive cells (in cortex, white matter, hippocampus, thalamus and striatum) and inhibited microglial cell activation in the thalamus. SN has neuroprotective potential in neonatal brain injury. Its main action seems to be inhibition of apoptosis in the aftermath of the insult, predominantly in the hypoxic-only hemisphere. This might be explained by the less pronounced injury in this hemisphere, where blood flow and thus nutrient supply are maintained.