Cerebral vascular and blood brain-barrier abnormalities in a mouse model of epilepsy and tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. METHODS: Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. In vivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. RESULTS: VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. SIGNIFICANCE: Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC.

A role of dentate gyrus mechanistic target of rapamycin activation in epileptogenesis in a mouse model of posttraumatic epilepsy.

OBJECTIVE: The mechanistic target of rapamycin (mTOR) pathway has been implicated in promoting epileptogenesis in animal models of acquired epilepsy, such as posttraumatic epilepsy (PTE) following traumatic brain injury (TBI). However, the specific anatomical regions and neuronal populations mediating mTOR's role in epileptogenesis are not well defined. In this study, we tested the hypothesis that mTOR activation in dentate gyrus granule cells promotes neuronal death, mossy fiber sprouting, and PTE in the controlled cortical impact (CCI) model of TBI. METHODS: An adeno-associated virus (AAV)-Cre viral vector was injected into the hippocampus of Rptorflox/flox (regulatory-associated protein of mTOR) mutant mice to inhibit mTOR activation in dentate gyrus granule cells. Four weeks after AAV-Cre or AAV-vehicle injection, mice underwent CCI injury and were subsequently assessed for mTOR pathway activation by Western blotting, neuronal death, and mossy fiber sprouting by immunopathological analysis, and posttraumatic seizures by video-electroencephalographic monitoring. RESULTS: AAV-Cre injection primarily affected the dentate gyrus and inhibited hippocampal mTOR activation following CCI injury. AAV-Cre-injected mice had reduced neuronal death in dentate gyrus detected by Fluoro-Jade B staining and decreased mossy fiber sprouting by ZnT3 immunostaining. Finally, AAV-Cre-injected mice exhibited a decrease in incidence of PTE. SIGNIFICANCE: mTOR pathway activation in dentate gyrus granule cells may at least partly mediate pathological abnormalities and epileptogenesis in models of TBI and PTE. Targeted modulation of mTOR activity in this hippocampal network may represent a focused therapeutic approach for antiepileptogenesis and prevention of PTE.

Construction of Tissue-engineered Cartilage In Vivo from Microtia Chondrocytes After Transfection with Human VEGF165 Genes Mediated by a Recombinant Adeno-Associated Viral Vector.

OBJECTIVE: To evaluate the transfection efficiency of cultured chondrocytes from individuals with microtia (microtia chondrocytes) with the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP and hVEGF165 in vitro. To test whether VEGF165 gene-modified microtia chondrocytes can enhance the survival and quality of tissue-engineered cartilage. METHOD: The recombinant plasmid rAAV2-hVEGF165-IRES-EGFP was inserted into rAAV2 virus vectors to construct rAAV2-hVEGF165-IRES-EGFP using the AATMaxTM system. The second-passage microtia chondrocytes were divided into 3 groups in vitro: the Ctr group (without transfection), Exp1 group (transfected with rAAV2-IRES-EGFP), and Exp2 group (transfected with rAAV2-hVEGF165-IRES-EGFP). At 24 h, 48 h, 72 h and 7 d after transfection, cell viability was measured by MTT staining. Transfection efficiency was determined by the rate of fluorescence-positive cells. The mRNA expression of hVEG165 was detected by RT-PCR (reverse transcription PCR) and agarose gel electrophoresis, and the VEGF165 protein levels in the supernatant fluids were measured by ELISAs. The second passage microtia chondrocytes with (Exp) and without (Ctr) transfection of VEGF165 genes were mixed with 0.5 ml 30% Pluronic F-127 at 4 °C and then injected subcutaneously into the opposing side of the back of nude mice. Eight weeks after injection, the cartilage-like tissues of nude mice were harvested for morphological and histologic examination. RESULTS: Chondrocyte viability increased in a time-dependent manner but did not differ among the 3 groups at the same time point. The mRNA and protein levels of VEGF increased in a time-dependent manner in the 3 groups. The mRNA and protein levels of VEGF165 were much higher in the Exp 2 group than in the Ctr and Exp 1 groups at the same time point, but the levels were not significantly different between the Exp 1 and Exp 2 groups. Both the Ctr group and the Exp1 group formed mature cartilage with mature lacunar structures, metachromatic matrices, collagen, and elastic fibers, and the structure of neonatal cartilage was not significantly different between the 2 groups. However, the wet weight of the neonatal cartilage was much larger in the Exp group (127.4 ± 12.4 mg) than in the Ctr group (58.5 ± 12.2 mg, p < 0.05). VEGF protein staining also showed a higher level in the Exp group. CONCLUSION: The HVEGF165 gene was transfected efficiently into microtia chondrocytes using the recombinant adeno-associated virus vector rAAV2-hVEGF165-IRES-EGFP. After transfection, the mRNA and protein levels of hVEGF165 increased in a time-dependent manner. VEGF165 gene-modified microtia chondrocytes showed enhanced survival in vivo but did not improve the texture of tissue-engineered cartilage. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description ofthese Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Hypothalamic orexin and mechanistic target of rapamycin activation mediate sleep dysfunction in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disease related to hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and manifested by neurological symptoms, such as epilepsy and sleep disorders. The pathophysiology of sleep dysfunction is poorly understood and is likely multifactorial, but may involve intrinsic biological regulators in the brain. Here, we characterized a mouse model of sleep disorders in TSC and investigated mechanisms of sleep dysfunction in this conditional knockout model involving inactivation of the Tsc1 gene in neurons and astrocytes (Tsc1GFAPCKO mice). Sleep studies utilizing EEG, EMG, and behavioral analysis found that Tsc1GFAPCKO mice have decreased REM sleep and impaired sleep-wake differentiation between light and dark phases. mTOR activity and orexin expression were increased in hypothalamic sections and cultured hypothalamic neurons from Tsc1GFAPCKO mice. Both the sleep abnormalities and increased orexin expression in Tsc1GFAPCKO mice were reversed by rapamycin treatment, indicating their dependence on mTOR activation. An orexin antagonist, suvorexant, also restored normal REM levels in Tsc1GFAPCKO mice. These results identify a novel mechanistic link between mTOR and orexin in the hypothalamus related to sleep dysfunction and suggest a targeted therapeutic approach to sleep disorders in TSC.

Comprehensive evaluation of environmental and economic benefits of China's urban underground transportation construction projects.

Urban underground transportation projects are introduced to address problems of scarce green land and traffic pollution. As construction of urban underground transportation is still in its infancy, there is no definite quantitative measurement on whether the construction is beneficial and what influences it will place on the region in China. This study intends to construct a comprehensive evaluation method for evaluating social, economic and environmental benefits of urban underground transportation projects and proposes the concept, role and principle for evaluation of environmental and economic benefits. It figures out relationship between the environment and factors of city development. It also summarizes three relevant factors, including transportation, biophysics and social economy, and works out indicators to evaluate the influence of urban underground transportation construction. Based on Contingent Valuation Method (CVM), Cost of Illness Approach (CIA), Human Capital Approach (HCA), this paper constructs 13 monetization calculation models for social, economic and environmental benefits in response to seven aspects, namely, reducing noise pollution and air pollution, using land efficiently, improving traffic safety, reducing traffic congestion, saving shipping time and minimizing transportation costs.

Modelling the assembly and flexibility of antibody structures.

Antibodies are large protein assemblies capable of both specifically recognising antigens and engaging with other proteins and receptors to coordinate immune action. Traditionally, structural studies have been dedicated to antibody variable regions, but efforts to determine and model full-length antibody structures are emerging. Here we review the current knowledge on modelling the structures of antibody assemblies, focusing on their conformational flexibility and the challenge this poses to obtaining and evaluating structural models. Integrative modelling approaches, combining experiments (cryo-electron microscopy, mass spectrometry, etc.) and computational methods (molecular dynamics simulations, deep-learning based approaches, etc.), hold the promise to map the complex conformational landscape of full-length antibody structures.

Influence of a Meltblown Die with a Laval Airstream Channel on the Manufacturing Process of a Polymer Fiber Based on an Orthogonal Test and Simulation Analysis.

A Laval nozzle is a device that accelerates a low-speed airstream to form a high-speed airstream. In this work, we use a Laval nozzle in the airstream channel design of a meltblown die to improve the tensile properties of the fiber in the airstream field of the meltblown die. The features of the airstream field of the meltblown die are analyzed by numerical simulation. For a given parametrization, six factors may be tuned to optimize the performance of the Laval airstream channel of the meltblown die. We thus use a five-level, six-factor orthogonal test method to optimize the airstream channel of the meltblown die to determine the various factors that influence the airstream field beneath the meltblown die. The results show that the optimized Laval meltblown die performs better than the traditional die and that the widths of the larynx and expansion segment most strongly affect the airstream velocity beneath the Laval meltblown die. Compared with a traditional die, the Laval die optimized by orthogonal testing increases the peak airstream velocity by 17.54%, average velocity by 96.81%, average temperature by 12.32%, and peak pressure by 14.61% and produces weaker turbulence intensity near the spinneret. These characteristics make the airstream beneath the die more stable and uniform and accelerate the attenuation of the fiber diameter, producing more polymer nanofibers. These results demonstrate a valuable approach to the design and optimization of meltblown dies and provide a technical reference for the production and application of the meltblown fiber production equipment.

Brief seizures cause dendritic injury.

Seizures may directly cause brain injury by disrupting the structure and function of synapses. Previous studies using in vivo time-lapse imaging have demonstrated an acute beading of dendrites and loss of dendritic spines immediately following status epilepticus, but the effects of brief seizures and the long-term evolution of this dendritic injury are unknown. Here, we examined the effects of seizures of varying durations on dendritic structure over several weeks using in vivo multiphoton imaging with kainate-induced seizures in mice. The degree of dendritic injury was directly dependent on the duration of the seizures, with seizures lasting more than 30 min (status epilepticus) resulting in a greater than 75% spine loss. However, even brief seizures (<5 min) induced moderate dendritic beading and spine loss. The dendritic injury from brief seizures usually recovered within 2 weeks, whereas status epilepticus-induced injury only partially reversed. These studies demonstrate that seizures of all durations may trigger at least transient neuronal injury.

Rapamycin Attenuates Acute Seizure-induced Astrocyte Injury in Mice in Vivo.

Astrocytes have been implicated in epileptogenesis and seizure-induced brain injury. Pathological studies reveal a variety of structural abnormalities in astrocytes, such as vacuolization and astrogliosis. While in vivo imaging methods have demonstrated rapid changes in astrocytes under a variety of physiological and pathological conditions, the acute effects of seizures on astrocyte morphology in vivo and corresponding mechanisms of seizure-induced astrocytic injury have not been documented. In this study, we utilized in vivo two-photon imaging to directly monitor the acute structural effects of kainate-induced seizures on cortical astrocytes. Kainate seizures cause an immediate, but transient, vacuolization of astrocytes, followed over several days by astrogliosis. These effects are prevented by pre- or post-treatment with rapamycin, indicating the mTOR pathway is involved in mediating seizure-induced astrocyte injury. These finding have clinical implications for mechanisms of seizure-induced astrocyte injury and potential therapeutic applications with mTOR inhibitors.

In Vivo Two-Photon Imaging of Astrocytes in GFAP-GFP Transgenic Mice.

Astrocytes play important roles in normal brain function and neurological diseases. In vivo two-photon excitation laser scanning microscopy has the potential to reveal rapid, dynamic structural changes in cells in a variety of physiological and pathological conditions. The type of in vivo imaging method has been shown to affect the plasticity of dendritic spines of neurons, but the optimal in vivo imaging methods of astrocytes have not been established. We compared open-skull and thinned-skull imaging methods for two-photon laser microscopy of live astrocytes in neocortex of GFAP-GFP transgenic mice. The thinned-skull method provided stable image intensity and morphological features of astrocytes in vivo over at least one week, with no evidence of astrogliosis. In contrast, the open-skull method resulted in significant changes in image intensity and induced astrogliosis. The thinned-skull method is the preferred approach for in vivo imaging of astrocytes under most conditions involving gross astrocyte modulation or causing astrogliosis.

Rapamycin prevents acute dendritic injury following seizures.

OBJECTIVE: Seizures cause acute structural changes in dendrites, which may contribute to cognitive deficits that occur in epilepsy patients. Disruption of the actin cytoskeleton of dendrites likely mediates the structural changes following seizures, but the upstream signaling mechanisms activated by synchronized physiological activity to cause seizure-induced dendritic injury are not known. In this study, we test the hypothesis that the mechanistic target of rapamycin complex 1 (mTORC1) pathway triggers structural changes in dendrites in response to seizures. METHODS: In vivo multiphoton imaging was performed in transgenic mice expressing green fluorescent protein in cortical neurons. The effect of rapamycin pre- and posttreatment was tested on kainate-induced dendritic injury and cofilin-mediated actin depolymerization. RESULTS: Kainate-induced seizures caused acute activation of mTORC1 activity, which was prevented by the mTORC1 inhibitor, rapamycin. Rapamycin pretreatment, and to a lesser degree, posttreatment attenuated acute seizure-induced dendritic injury and correspondingly decreased LIM kinase and cofilin-mediated depolymerization of actin. INTERPRETATION: The mTORC1 pathway mediates seizure-induced dendritic injury via depolymerization of actin. These findings have important mechanistic and translational applications for management of seizure-induced brain injury.

Rapamycin attenuates the development of posttraumatic epilepsy in a mouse model of traumatic brain injury.

Posttraumatic epilepsy is a major source of disability following traumatic brain injury (TBI) and a common cause of medically-intractable epilepsy. Previous attempts to prevent the development of posttraumatic epilepsy with treatments administered immediately following TBI have failed. Recently, the mammalian target of rapamycin complex 1 (mTORC1) pathway has been implicated in mechanisms of epileptogenesis and the mTORC1 inhibitor, rapamycin, has been proposed to have antiepileptogenic effects in preventing some types of epilepsy. In this study, we have tested the hypothesis that rapamycin has antiepileptogenic actions in preventing the development of posttraumatic epilepsy in an animal model of TBI. A detailed characterization of posttraumatic epilepsy in the mouse controlled cortical impact model was first performed using continuous video-EEG monitoring for 16 weeks following TBI. Controlled cortical impact injury caused immediate hyperactivation of the mTORC1 pathway lasting at least one week, which was reversed by rapamycin treatment. Rapamycin decreased neuronal degeneration and mossy fiber sprouting, although the effect on mossy fiber sprouting was reversible after stopping rapamycin and did not directly correlate with inhibition of epileptogenesis. Most posttraumatic seizures occurred greater than 10 weeks after TBI, and rapamycin treatment for one month after TBI decreased the seizure frequency and rate of developing posttraumatic epilepsy during the entire 16 week monitoring session. These results suggest that rapamycin may represent a rational treatment for preventing posttraumatic epilepsy in patients with TBI.

Video-EEG monitoring methods for characterizing rodent models of tuberous sclerosis and epilepsy.

Tuberous Sclerosis Complex (TSC) is a genetic disease involving dysregulation of the mTOR pathway and resulting in disabling neurological manifestations, such as epilepsy. Animal models may recapitulate epilepsy and other behavioral features of TSC and are useful tools for investigating mechanisms of epileptogenesis and other neurological deficits in TSC. In this chapter, methods for performing video-electroencephalography (video-EEG) to characterize epilepsy and neurological dysfunction in rodent models are reviewed. In particular, technical aspects of surgical implantation of EEG electrodes, video-EEG recording, and analysis and interpretation of EEG data are detailed. These methodological approaches should be helpful in characterizing seizures and background EEG abnormalities not only in animal models of TSC but also in many rodent epilepsy models in general.

Clinical analysis, repair and aetiology of question mark ear.

UNLABELLED: The question mark ear is a malformation involving a cleft between the helix and the earlobe. The upper portion of the ear appears protruding and the scapha is absent from the affected region. The severity of this ear malformation varies from a small notch in the helix to complete separation of the helix and the lobe. In this article, we classify the question mark ear according to the severity of the malformation and propose two different methods of correction. In addition, we review the aetiology of the question mark ear and hope this can enhance the understanding of the pathology. METHODS: In the repair of moderate question mark ears, the local chondrocutaneous flap is used. In severe cases, tissue expander and autogenous rib cartilage are applied to reconstruct the deficiency of the lower part of the ear. RESULTS: There were 32 cases of question mark ears from July 2003 to December 2007. Thirty cases were sporadic and two cases had familial history. Twenty-two moderate question mark ears were repaired using chondrocutaneous flap transposition and ten severe question mark ears were repaired using tissue expander and autogenous cartilage. CONCLUSIONS: In moderate question mark ears, the corrected auricle showed a shape similar to that of the unaffected auricle, but the size was a little smaller. Most of the reconstructed severe question mark ears showed excellent contour.

Fabrication of three-dimensional cartilaginous framework in auricular reconstruction.

OBJECTIVE: One of the greatest challenges in facial plastic surgery is total auricular reconstruction, as it demands precise technique as well as artistic creativity. The crucial step to reconstruct satisfactory auricles is sculpting a three-dimensional cartilaginous framework, which determines the success of surgery. In this paper we describe our new techniques in fabricating the three-dimensional auricular framework. METHODS: To assemble satisfactory auricular frameworks, we designed four different methods according to the number of rib cartilages used. (1) Method I, the framework was made from the entire 6th, 7th and 8th rib cartilages; (2) method II, the framework was made from the partial 6th, the entire 7th and 8th rib cartilages; (3) method III, the framework was made from the entire 7th and 8th rib cartilages; (4) method IV, the framework was made only from the entire 7th rib cartilage. RESULTS: 332 patients, ranging in age from 5 to 33 years old, were operated on using autogenous costal cartilage between 2004 and 2006. All the reconstructed ears had a satisfactory three-dimensional configuration. The cranioauricular angle of the reconstructed ears was also similar to that of the opposite ears. CONCLUSIONS: According to the status of the normal ear and the development of the cartilage in each patient, we have developed four methods to harvest and fabricate cartilage. Our operations on 332 patients with microtia suggest that our new technique is effective in fabricating cartilaginous framework. Our new technique facilitates the operation procedure and minimises the usage of cartilage.

[Reconstruction of contracted eye socket using expanded forehead island skin flap].

OBJECTIVE: To investigate the feasibility of reconstruction of the contracted eye socket by an application of the expanded forehead island skin flap with the supratrochlear and supraorbital arteries. METHODS: From June 2002 to June 2005, 6 patients with the eye socket defects were treated with an expanded forehead island skin flap with the supratrochlear and supraorbital arteries. There were 4 males and 2 females, aged 16-42 years. The defects were caused by tumors in 2 patients, by trauma in 3, and by chemical burns in 1; the defects were in the left eyes of 4 patients and in the right eyes of the remaining 2 patients, with the illness course of 1 year to 4 years. All the patients first underwent the skin and soft tissue expanding operation on the donor forehead skin area; 1 month later, the transplant of the expanded forehead island skin flap with the supratrochlear and supraorbital arteries was performed to reconstruct the eye sockets. The flaps ranged in size from 8 cm x 5 cm to 10 cm x 6 cm. The appearance and functional recovery of the reconstructed eye sockets were observed after operation. RESULTS: The follow-up of all the patients for 1-3 years revealed that the skin flaps survived, with no visible contracture, and the fine sensory function was still present. The artificial eyes could be steadily placed in the reconstructed eye sockets. The donor areas were healed with no visible hyperplastic scars. CONCLUSION: Reconstruction of the eye socket with an expanded forehead island skin flap with the supratrochlear and supraorbital arteries is a feasible, effective and simply method, and the patient can have a concealed incision, a satisfactory appearance, and a fine sensory function.

[Clinical significance of vascular distribution type of the expanded skin in the mastoid region in congenital microtia].

OBJECTIVE: To classify the blood vessel distribution of the expanded skin in the mastoid region and its relevance to reasonable and reliable design of the expanded flap for auricular reconstruction in congenital microtia. METHODS: The blood vessel distribution of the expanded skin in the mastoid region was observed by light permeation test. The expanded flaps with different ratio of length to width were designed according to their blood vessel distribution types. RESULTS: The vascular distribution of the expanded skin in 403 cases was divided into five types. All the flaps survived completely. CONCLUSIONS: The blood vessel distribution type of the expanded skin in mastoid region has great significance for the design of post-auricular expanded flap in auricular reconstruction.

[Surgical treatment of acquired ear defect].

OBJECTIVE: To explore surgical procedure for the treatment of acquired ear defect. METHODS: The ear reconstruction was carried out by using soft tissue skin expander and autogenous rib cartilage framework, Medpor framework, auricular prosthesis, and so on. RESULTS: The long-term follow-ups showed that the flap of reconstructed ear was ruddy, soft, with normal sensory function. The cartilage framework had no degeneration, absorption and deformation. The Medpor framework had no deformation. The contour of auricular prosthesis was vivid and the implant system was stable. In addition, the reconstructed ears were coincidence with the normal side on location, form and dimension. CONCLUSIONS: It may be a major method for traumatic ear reconstruction that the soft tissue skin expander with autogenous rib cartilage framework could be applied at present time. In some special circumstances, the soft tissue skin expander with Medpor framework and auricular prosthesis may be other helpful choices.

[Classification and repairment of "butterfly ear" deformity].

OBJECTIVE: To classify and repair "Butterfly Ear" deformity which presents characters of dysplasia of inferior auricle of ear and congenital bat ear. METHOD: The repairment procedures include: type I: auricular cartilage flap inversion folding technique. type II: local ear skin flap. type III: soft tissue expander autogenous, rib cartilage framework. RESULTS: The method was used in 19 cases from October 2001 to March 2005. Postoperative follow-up showed satisfactory results in all cases. CONCLUSION: According to "Butterfly Ear" deformity classification, different technique could be applied.