Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.     

利拉鲁肽抑制高糖诱导的小鼠胰岛细胞损伤

利拉鲁肽(liraglutide, Lira)是胰高血糖素样肽-1的类似物,在糖尿病治疗中发挥重要作用,但利拉鲁肽通过改善胰岛β细胞的功能实现治疗糖尿病的具体机制尚未完全阐明。本研究采用高糖(33 mmol/L)诱导胰岛MIN6细胞48 h建立高糖损伤模型,CCK-8检测发现,与对照组相比,高糖组MIN6细胞活力下降(P<0.05),利拉鲁肽作用高糖组细胞活力升高(P<0.05);小鼠胰岛素和ATP含量检测发现,与对照组相比,高糖组胰岛素分泌降低(P<0.01),ATP含量减少(P<0.001),利拉鲁肽作用高糖组胰岛素释放量增加(P<0.05)和细胞内ATP含量增加(P<0.001);采用活体细胞线粒体膜通道孔(MPTP)荧光检测发现,与对照组相比,高糖组绿色荧光强度降低(P<0.001),利拉鲁肽作用高糖组绿色荧光强度增加(P<0.001);DCFH-DA探针联合流式细胞仪检测细胞活性氧簇(ROS)含量发现,与对照组相比,高糖组ROS水平升高(P<0.001),利拉鲁肽作用高糖组ROS水平降低(P<0.01);细胞内丙二醛...     

Synapse Loss and Dendrite Remodeling in a Mouse Model of Glaucoma

It has been hypothesized that synaptic pruning precedes retinal ganglion cell degeneration in glaucoma, causing early dysfunction to retinal ganglion cells. To begin to assess this, we studied the excitatory synaptic inputs to individual ganglion cells in normal mouse retinas and in retinas with ganglion cell degeneration from glaucoma (DBA/2J), or following an optic nerve crush. Excitatory synapses were labeled by AAV2-mediated transfection of ganglion cells with PSD-95-GFP. After both insults the linear density of synaptic inputs to ganglion cells decreased. In parallel, the dendritic arbors lost complexity. We did not observe any cells that had lost dendritic synaptic input while preserving a normal or near-normal morphology. Within the temporal limits of these observations, dendritic remodeling and synapse pruning thus appear to occur near-simultaneously.     

Sodium Ferulate Inhibits Neointimal Hyperplasia in Rat Balloon Injury Model

Background/Aim

Neointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved.

Methods

Cultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed.

Results

In presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group.

Conclusion

Sodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs.     

Injury, Endogenous Starch Production, and Nucleolar Enlargement in Lemon Fruit Juice Vesicles

Excision of lemon fruit juice vesicles from their natural connectionwith the fruit wall is the minimal physical stimulus neededfor initiating the physiological processes which lead to endogenousstarch production in this tissue system. Endogenous starch formationoccurs in the juice vesicle cells over a wide range of environmentalconditions and takes place concomitantly with the phenomenonof nucleolar enlargement.     

柑桔果汁中苦味物质的去除方法

类柠檬苦素和柚皮苷是柑桔果汁中的主要苦味成分,会明显的降低柑桔果汁的品质。本文综述了柑桔果汁的苦味机理,苦味物质的去除方法。     

变应性鼻炎模型小鼠鼻黏膜的形态学观察

目的:建立小鼠变应性鼻炎模型,观察小鼠鼻腔黏膜组织的重塑情况。方法:20只BALB/c小鼠被随机分为致敏组和对照组,使用卵清蛋白(OVA)诱导建立小鼠变应性鼻炎模型。通过HE染色观察小鼠鼻黏膜的大体重塑情况,吉姆萨染色观察嗜酸性粒细胞,阿辛蓝-过碘酸-希夫染色观察杯状细胞;酶联免疫吸附(ELISA)法检测小鼠血清中白细胞介素-4(IL-4)的水平。结果:小鼠变应性鼻炎模型的生物学行为评分为6.5±1.3,提示造模成功。与对照组相比,致敏组鼻腔黏膜出现上皮细胞脱落、坏死,杯状细胞增生,鳞状上皮化生,固有层和黏膜下层腺体增生、血管扩张,组织水肿,固有层内可见特征性的嗜酸性粒细胞浸润,造模后鼻腔黏膜结构存在重塑。致敏组小鼠鼻黏膜嗜酸性粒细胞计数及杯状细胞计数分别为(26.4±5.72)和(24.14±3.12),而对照组分别是(8.31±2.42)和(9.41±1.22),两组比较均具有统计学差异(P0.05);致敏组血清中白细胞介素4(IL-4)水平为(18.9±3.1)pg/ml,对照组为(8.3±1.4)pg/ml,致敏组显著高于对照组,差异有统计学意义(P0.05)。结论:通过卵清蛋白诱导建立的小鼠变应性鼻炎模型鼻腔黏膜存在组织重塑。     

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.     

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.     

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called ‘angiogenic privilege'' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.     

Mouse Model of Muscle Crush Injury of the Legs

Because crush injury to skeletal muscle is an important cause of morbidity in natural disaster and battlefield settings, a reproducible and refined animal model of muscle crush injury is needed. Both open and closed small-animal models of skeletal muscle crush injury are available but are limited by their need for surgical isolation of the muscle or by the adverse effect of fibular fracture, respectively. In the current study, we developed and validated a novel, noninvasive mouse model of lower-extremity muscle crush injury. Despite the closed nature of our model, gross evidence of muscle damage was evident in all mice and was verified microscopically through hematoxylin and eosin staining. The injury elicited both neutrophil and macrophage infiltration at 24 and 48 h after injury. The area percentage and mean antigen area of F4/80-positive macrophages were higher at 48 h than at 24 h after injury, and CD68-positive macrophage area percentage and mean antigen area differed significantly between injured and uninjured muscle. In addition, the incidence of fibular fracture was one third lower than that reported for an alternative noninvasive model. In conclusion, our model is a reproducible method for muscle crush injury in the mouse pelvic limb and is a refinement of previous models because of its decreased bone fractures and reduction of animal numbers.Abbreviations: AOI, area of interestSkeletal muscle crush injury is an important cause of morbidity in both civilian and military populations. During earthquakes, tornados, and other natural disasters, collapsed structures result in crush injuries in approximately 40% of victims entrapped in the rubble,¹⁴ and crush injuries sustained during these events primarily affect skeletal muscle tissue.¹³ Crush injuries to skeletal muscle received during military conflict can occur when a limb is compressed for an extended time period, and combat-related crush injuries of the extremities frequently are present in wounded troops who are transported via aeromedical evacuation.²²A muscle-crush injury is induced when pressure is applied to skeletal muscle, interrupting blood flow and damaging the cell membranes of the muscle fibers. Several animal models of skeletal muscle-crush injury are used to study the pathophysiology of acute muscle inflammation and to investigate potential therapies.^{1,2,3,5,8,9,12,17,18,21} The most common model is the application of force to a surgically isolated pelvic limb muscle by using a clamp.¹⁶ Although closed models have been investigated, these studies typically involve dropping weights onto rodents’ pelvic limbs, thereby increasing the adverse event of fractures. Although not often reported in the literature, the incidence of fibular fractures in rats as a result of the dropped weight was 27% in one study.³ An additional drawback to the dropped-weight model is that it simulates a high-force contusion injury and does not provide the ischemic effect of the continuous pressure applied by the open clamp model. The ideal crush-injury model would mimic a force-induced injury, because 40% of survivors trapped in building rubble develop ischemia-induced crush syndrome.¹⁴We chose to investigate a novel model of closed crush injury for several reasons. An animal model of skeletal muscle injury should mimic the human clinical presentation, and a closed model more closely simulates a real-world crush injury. Second, because the incision created in the open model can activate the inflammatory response, a group of sham-operated animals is needed to control for the variable of the incision-induced inflammation. By using a closed model, the contralateral limb can serve as the uninjured control, thereby reducing the number of animals needed to perform the study. Last, the closed model represents a refinement of the crush injury procedure by removing the additional tissue damage and inflammation that result from the incision and tissue dissection of the surgical procedure and by reducing the incidence of fractures.Because this model has not been described in the literature, the objective of the current study was to develop a closed, sustained-force model of lower-extremity crush injury that induces a measurable leukocyte response and minimizes damage to nearby bones. In addition, we used monoclonal antibodies to characterize the leukocyte populations associated with this skeletal muscle crush injury model.     

Intermittent Fasting Attenuates Hallmark Vascular and Neuronal Pathologies in a Mouse Model of Vascular Cognitive Impairment

Background - Chronic cerebral hypoperfusion (CCH) is an important pathophysiological mechanism of vascular cognitive impairment (VCI). The heterogeneous effects of CCH complicate establishing single target therapies against VCI and its more severe form, vascular dementia (VaD). Intermittent fasting (IF) has multiple targets and is neuroprotective across a range of disease conditions including stroke, but its effects against CCH-induced neurovascular pathologies remain to be elucidated. We therefore assessed the effect of IF against CCH-associated neurovascular pathologies and investigated its underlying mechanisms.Methods - Male C57BL/6NTac mice were subjected to either ad libitum feeding (AL) or IF (16 hours of fasting per day) for 4 months. In both groups, CCH was experimentally induced by the bilateral common carotid artery stenosis (BCAS) method. Sham operated groups were used as controls. Measures of leaky microvessels, blood-brain barrier (BBB) permeability, protein expression of tight junctions, extracellular matrix components and white matter changes were determined to investigate the effect of IF against CCH-induced neurovascular pathologies.Results - IF alleviated CCH-induced neurovascular pathologies by reducing the number of leaky microvessels, BBB breakdown and loss of tight junctional proteins. In addition, IF mitigated the severity of white matter lesions, and maintained myelin basic protein levels, while concurrently reducing hippocampal neuronal cell death. Furthermore, IF reduced the CCH-induced increase in levels of matrix metalloproteinase (MMP)-2 and its upstream activator MT1-MMP, which are involved in the breakdown of the extracellular matrix that is a core component of the BBB. Additionally, we observed that IF reduced CCH-induced increase in the oxidative stress marker malondialdehyde, and increased antioxidant markers glutathione and superoxide dismutase. Overall, our data suggest that IF attenuates neurovascular damage, metalloproteinase and oxidative stress-associated pathways, and cell death in the brain following CCH in a mouse model of VCI.Conclusion - Although IF has yet to be assessed in human patients with VaD, our data suggest that IF may be an effective means of preventing the onset or suppressing the development of neurovascular pathologies in VCI and VaD.     

Plasminogen Activators in Vascular Remodeling and Angiogenesis

This review considers cellular and molecular mechanisms of the involvement of plasminogen activators in extracellular proteolysis and cell migration and proliferation. The role of plasminogen activators in vascular remodeling in atherosclerosis, restenosis, and angiogenesis is discussed.     

Hypertonic Saline Reduces Vascular Leakage in a Mouse Model of Severe Dengue

Dengue (DEN) is a mosquito-borne viral disease and represents a serious public health threat and an economical burden throughout the tropics. Dengue clinical manifestations range from mild acute febrile illness to severe DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS). Currently, resuscitation with large volumes of isotonic fluid remains the gold standard of care for DEN patients who develop vascular leakage and shock. Here, we investigated the ability of small volume of hypertonic saline (HTS) suspensions to control vascular permeability in a mouse model of severe DEN associated with vascular leakage. Several HTS treatment regimens were considered and our results indicated that a single bolus of 7.5% NaCl at 4 mL per kg of body weight administered at the onset of detectable vascular leakage rapidly and significantly reduced vascular leak for several days after injection. This transient reduction of vascular leakage correlated with reduced intestine and liver damage with restoration of the hepatic functions, and resulted in delayed death of the infected animals. Mechanistically, we showed that HTS did not directly impact on the viral titers but resulted in lower immune cells counts and decreased systemic levels of soluble mediators involved in vascular permeability. In addition, we demonstrated that neutrophils do not play a critical role in DEN-associated vascular leakage and that the therapeutic effect of HTS is not mediated by its impact on the neutrophil counts. Together our data indicate that HTS treatment can transiently but rapidly reduce dengue-associated vascular leakage, and support the findings of a recent clinical trial which evaluated the efficacy of a hypertonic suspension to impact on vascular permeability in DSS children.     

Vismodegib Suppresses TRAIL-mediated Liver Injury in a Mouse Model of Nonalcoholic Steatohepatitis

Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.     

Emodin Inhibits Inducible Nitric Oxide Synthase in a Rat Model of Craniocerebral Explosive Injury

To investigate the effects of emodin on blast-induced traumatic brain injury (bTBI) in a rat model. Eighty rats were randomly divided into 2 groups (the control group and the emodin-treated group; N = 40 per group) and were used to establish the model of blast-induced traumatic brain injury. Ten minutes after the explosion, an isotonic saline solution (10 mg/kg) or emodin (10 mg/kg) were administered via an intraperitoneal injection to the control group and the emodin-treated group, respectively. At each time point (pre-explosion, 2, 6, 12, 24 h after explosion), 2 rats were used for the pathological assessment and 6 rats were used for the biochemical assessment. The concentration of nitric oxide (NO) and the expression and activity of inducible nitric oxide synthase (iNOS) were measured at each time point by spectrophotometry and western blot analysis. Light and electron microscopy showed that the brain damage in the emodin-treated group was less serious than that observed in the control group. The concentration of NO in the emodin-treated group was lower compared with the control group (p < 0.05). Western blot analysis showed that protein expression in the emodin-treated group was lower than the control group (p < 0.05). Emodin can alleviate brain damage after bTBI by inhibiting iNOS. These findings suggest that emodin has a protective effect against bTBI. One possible mechanism may occur by inhibiting the expression and activity of iNOS and consequently decreasing the concentration of NO.     

Vigabatrin Inhibits Seizures and mTOR Pathway Activation in a Mouse Model of Tuberous Sclerosis Complex

Epilepsy is a common neurological disorder and cause of significant morbidity and mortality. Although antiseizure medication is the first-line treatment for epilepsy, currently available medications are ineffective in a significant percentage of patients and have not clearly been demonstrated to have disease-specific effects for epilepsy. While seizures are usually intractable to medication in tuberous sclerosis complex (TSC), a common genetic cause of epilepsy, vigabatrin appears to have unique efficacy for epilepsy in TSC. While vigabatrin increases gamma-aminobutyric acid (GABA) levels, the precise mechanism of action of vigabatrin in TSC is not known. In this study, we investigated the effects of vigabatrin on epilepsy in a knock-out mouse model of TSC and tested the novel hypothesis that vigabatrin inhibits the mammalian target of rapamycin (mTOR) pathway, a key signaling pathway that is dysregulated in TSC. We found that vigabatrin caused a modest increase in brain GABA levels and inhibited seizures in the mouse model of TSC. Furthermore, vigabatrin partially inhibited mTOR pathway activity and glial proliferation in the knock-out mice in vivo, as well as reduced mTOR pathway activation in cultured astrocytes from both knock-out and control mice. This study identifies a potential novel mechanism of action of an antiseizure medication involving the mTOR pathway, which may account for the unique efficacy of this drug for a genetic epilepsy.     

Angiogenesis and Vascular Remodeling in Chronic Airway Diseases

Asthma and chronic obstructive pulmonary disease remain a global health problem, with increasing morbidity and mortality. Despite differences in the causal agents, both diseases exhibit various degrees of inflammatory changes, structural alterations of the airways leading to airflow limitation. The existence of transient disease phenotypes which overlap both diseases and which progressively decline the lung function has complicated the search for an effective therapy. Important characteristics of chronic airway diseases include airway and vascular remodeling, of which the molecular mechanisms are complex and poorly understood. Recently, we and others have shown that airway smooth muscle (ASM) cells are not only structural and contractile components of airways, rather they bear capabilities of producing large number of pro-inflammatory and mitogenic factors. Increase in size and number of blood vessels both inside and outside the smooth muscle layer as well as hyperemia of bronchial vasculature are contributing factors in airway wall remodeling in patients with chronic airway diseases, proposing for the ongoing mechanisms like angiogenesis and vascular dilatation. We believe that vascular changes directly add to the airway narrowing and hyper-responsiveness by exudation and transudation of proinflammatory mediators, cytokines and growth factors; facilitating trafficking of inflammatory cells; causing oedema of the airway wall and promoting ASM accumulation. One of the key regulators of angiogenesis, vascular endothelial growth factor in concerted action with other endothelial mitogens play pivotal role in regulating bronchial angiogenesis. In this review article we address recent advances in pulmonary angiogenesis and remodelling that contribute in the pathogenesis of chronic airway diseases.