Increased Expression of Rac1 in Epilepsy Patients and Animal Models

The mechanisms of epilepsy remain incompletely understood. Rac1 (ras-related C3 botulinum toxin substrate 1) belongs to the Rho family of small GTPases. Rac1 play important roles in cytoskeleton rearrangement and neuronal synaptic plasticity, which had also been implicated in epilepsy. However, little is known regarding the expression of Rac1 in the epileptic brain or whether Rac1-targeted interventions affect the progression of epilepsy. The aim of this study was to investigate the expression profile of Rac1 in brain tissues from patients suffering from temporal lobe epilepsy (TLE) and experimental epileptic rats and determine the possible role of Rac1 in epilepsy. We demonstrated that the expression of Rac1 is significantly increased in TLE patients and in lithium-pilocarpine epilepsy model animals compared to the corresponding controls. Rac1 inhibitor NSC23766 reduced the severity of status epilepticus during the acute stage in a lithium-pilocarpine animal model. Consistent with these results, the latent period of a PTZ kindling animal model also increased. Our results demonstrated that the increased expression of Rac1 may contribute to pathophysiology of epilepsy.

     

临床抗癫痫新药治疗的动物实验

目的:对比临床较常用的几种抗癫痫新药与传统抗癫痫药苯妥英钠长期单药治疗慢性颞叶癫痫大鼠的疗效与安全性。方法:用海仁酸制成慢性颞叶癫痫的大鼠模型,临床常用维持剂量的苯妥因钠、奥卡西平、拉莫三嗪、妥泰根据成人剂量换算为大鼠剂量分别为37．50mg/Kg、125mg/Kg、28．13mg/Kg、15．63mg/Kg。每日灌胃,连续90天后,观察并比较各用药组大鼠脑电功率谱,学习记忆能力,血液常规,肝功能的变化情况。结果:90天后,与造模组相比,苯妥因钠、奥卡西平、拉莫三嗪、妥泰各组脑电活动均改善,其中以拉莫三嗪组最明显。水迷宫测试中:奥卡西平组较好;各用药组对造血、肝功能的影响与造模组及正常对照组相比无统计学意义。结论:对于慢性颞叶癫痫大鼠,拉莫三嗪改善脑电最明显,奥卡西平明显改善认知功能,疗效优于妥泰和苯妥英钠。     

Animal Models of Q Fever (Coxiella burnetii)

Q fever, caused by the pathogen Coxiella burnetii, is an acute disease that can progress to become a serious chronic illness. The organism leads an obligate, intracellular lifecycle, during which it multiplies in the phagolytic compartments of the phagocytic cells of the immune system of its hosts. This characteristic makes study of the organism particularly difficult and is perhaps one of the reasons why, more than 70 y after its discovery, much remains unknown about the organism and its pathogenesis. A variety of animal species have been used to study both the acute and chronic forms of the disease. Although none of the models perfectly mimics the disease process in humans, each opens a window onto an important aspect of the pathology of the disease. We have learned that immunosuppression, overexpression of IL10, or physical damage to the heart muscle in mice and guinea pigs can induce disease that is similar to the chronic disease seen in humans, suggesting that this aspect of disease may eventually be fully understood. Models using species from mice to nonhuman primates have been used to evaluate and characterize vaccines to protect against the disease and may ultimately yield safer, less expensive vaccines.Coxiella burnetii is the causative agent of human Q fever. Infection can take several forms and has been described as clinically polymorphic.⁶ In humans, presentation ranges from asymptomatic, through acute disease, to chronic illness. In the majority of cases, acute disease presents as a self-limiting febrile illness, with half of cases also having severe headaches.⁸⁸ In severe cases of acute disease, atypical pneumonia is often found.⁸⁸ A small proportion (2% to 4%) of subjects with symptomatic acute Q fever are admitted to hospital.^70,88 Chronic disease may develop in approximately 5% of those infected;¹⁶ the vast majority of these cases will present as a bacterial culture-negative endocarditis^16,22 often in those with predisposing heart-damage¹⁹ or immunosuppression.¹⁶ Without effective treatment, Q fever endocarditis is generally fatal, but early diagnosis coupled with novel treatment strategies has brought the death rate down to less than 5%.⁶⁹ The 2009 outbreak in the Netherlands involved 2357 human cases, of which more than 400 required hospitalization.⁹⁰ The animal cost in the Netherlands was far higher, with more than 50,000 pregnant goats culled in an attempt to control the epidemic.⁸²Two other clinical manifestations of Q fever are worthy of mention owing to their less-than-satisfactory outcomes with current treatment strategies. These are Q fever during pregnancy and Q fever fatigue syndrome. C. burnetii infection during pregnancy results in premature delivery in almost half of those affected and spontaneous abortion in more than a quarter.¹⁴ There have been few studies in this area, but there are indications that among those infected during the first trimester and treated suboptimally, the abortion rate is 100%.⁶⁸ This effect is compounded by the fact that the frontline bactericidal drugs for treatment (doxycycline and hydroxychloroquine) are contraindicated for use during pregnancy.⁶⁸ A bacteriostatic regimen (cotrimoxazole) has therefore been proposed for use⁶⁸ until delivery. Without satisfactory treatment during and after pregnancy, there is also a high probability for infection to lead to chronic Q fever: an incidence of 70% was reported in a group of pregnant women in France.⁶⁸Post-Q fever fatigue syndrome was first reported in 1996,⁵² but an association between Q fever and chronic fatigue had been observed as early as 1982.⁵² Between 10% and 15% of those who have had acute Q fever develop a chronic fatigue syndrome that can last between 5 and 10 y—and even longer in some cases.⁵³ Some of these patients have been found to have long-term persistence of C. burnetii cell components and LPS associated with traces of genomic DNA,⁵³ suggesting that Q fever fatigue syndrome may be immunologically mediated rather than caused by the organism directly.Q fever is a zoonosis that has been described worldwide,⁵⁶ and human outbreaks are often associated with contact with the birth products of farm animals.⁵⁶ However, outbreaks associated with the birth products of domestic cats have also been reported.⁵⁴ Human infection primarily occurs via the inhalation of infectious aerosols.⁵⁶ Over the past 10 y, outbreaks have been reported in the Netherlands,⁷¹ Slovenia,²⁶ the United Kingdom,^91,97,99 Israel,² Iraq,¹⁸ the United States,¹¹ Germany,²⁴ Bulgaria,⁶³ Croatia,⁵⁸ Spain,²³ Italy,⁸³ and France.⁸⁸A very small number of C. burnetii organisms can cause infection by inhalation. Infection has been predicted to be possible after exposure to only a single organism.³³ This low dosage, coupled with the organism''s ability to cause debilitating disease and high levels of resistance to various means of inactivation^67,77,78 have resulted in it being listed as a category B biologic warfare and bioterrorism agent by the Centers for Disease Control.⁴⁹Prevention of Q fever in man can be achieved by vaccination; the only vaccine available for general use is Q-Vax, which was licensed in Australia in 1989.⁵¹ This vaccine consists of formalin-inactivated C. burnetii whole cells, produced in chick embryos. Its use has been associated with severe local reactions in those with preexisting immunity. As a precaution, prevaccination screening (history, skin test, and serology) must therefore be performed prior to administration.³⁵ Despite this safeguard, severe local reactions to vaccination are reported.⁴⁴ The vaccine is also hazardous to produce, with the organism requiring culture in chick-embryos at biosafety level 3 prior to inactivation.⁵¹ There is, therefore, a need for a vaccine that is safer to produce and safer to use and that does not require prevaccination screening.The organism displays antigenic phase variation often paralleled with the rough-smooth variation seen in Enterobacteriaceae. In C. burnetii, phase variation has been demonstrated to be due to differences in LPS. Phase I has been shown to contain a unique disaccharide galactosaminuronyl glucosamine and 9 unidentified components in addition to the components of phase II LPS.¹ Organisms with the phase I phenotype are the infectious and virulent form found in the environment. Organisms with the phase II phenotype are observed only during repeated subculture in laboratory chick embryo or cell culture systems;²⁷ they have a chemically simpler LPS¹ and several deletions in the genome.^32,92 Phagocytosis of phase I, but not phase II, organisms by macrophages involves an interaction between the bacterial LPS and Toll-like receptor 4. This mechanism also stimulates F-actin reorganization of the host cells and stimulates the release of type 1 cytokines including IFNγ and TNF.³⁰ This interaction appears important in the initial priming of the immune response and could provide an explanation for the limited protection of vaccines based on potential virulence genes (omp1, HspB, Pmm, Fbp, Orf 410, Crc, CbMip, MucZ, P28) singly and in combinations but containing no LPS.^47,89,102In addition to its antigenic phase variation, C. burnetii occurs in 2 morphologic forms, a large-cell variant and a small-cell variant. These forms differ antigenically due to differences in the proteins expressed on their surface. It has been suggested that the resistance of C. burnetii to host defense mechanisms may be enhanced by antigenic differences between the different developmental forms.^57,94 The small-cell morphologic form is highly resistant to destruction by chemical and environmental factors and is likely the transmissible form of the pathogen.^15,67 After infection, which generally occurs by inhalation of the small-cell form, the organisms are taken up by host alveolar macrophages.⁸¹ Morphogenesis from the small-cell to large-cell form then occurs, the large-cell variant being the replicative form of the organism.¹⁵ These organisms then replicate within parasitophorous vacuoles.⁵⁰ As the organisms enter the stationary phase of their growth within the cell, they condense back into the small-cell form.¹⁵ During replication within the host cell, the organism subverts cellular processes though active mechanisms to avoid and modify the host immune response.⁵⁰ C. burnetii possesses a type IV secretion system, and the proteins that cause this subversion are likely delivered to the host cell by this machinery.^50,93Because C. burnetii is an obligate intracellular organism, it has only been possible to study the organism within living animal hosts. Host-cell–free growth of the organism has been reported recently,⁶² but the technique has yet to be exploited fully. Cell-culture–based in vitro systems remain limited in the study of C. burnetii, given that the organism soon reverts to the avirulent (at least in immunocompetent hosts) phase II form (characterized by the loss of the phase I LPS phenotype) in these systems.¹⁰ A key problem in comparing models of C. burnetii infection is related to the organism''s intracellular nature, which complicates attempts to count the organisms used for infection. The literature reflects this difficulty in the fact that there are many different methods used (including plaque assay in primary cell cultures, median infectious doses in chick eggs or mice, and median lethal dose in SCID mice) and no way to directly compare them.     

左旋组氨酸对匹罗卡品致痫大鼠癫痫发作和海马区神经细胞凋亡的影响

目的:观察左旋组氨酸(L-His)对匹罗卡品(PLO)致痫大鼠皮质脑电图及大鼠海马各区神经细胞凋亡的影响。方法:雄性SD大鼠30只,随机平均分为对照组(匹罗卡品组)、干预组(匹罗卡品+左旋组氨酸组)。各组给予相关干预处理后腹腔注射匹罗卡品建立癫痫模型,行皮质脑电图观察及海马区细胞凋亡染色观察。结果:经皮质脑电图显示,干预组潜伏期延长、痫波频率及大发作次数较对照组明显降低,差异有统计学意义(P<0.05)。凋亡染色显示,对照组的海马各区细胞在各时间点的凋亡数明显高于干预组,差异具有统计学意义(P<0.05)。结论:左旋组氨酸可以延迟匹罗卡品致痫的形成并降低点燃后癫痫发作程度,降低海马各区细胞凋亡数值,提示左旋组氨酸具有抗痫作用。     

Animal Models of Vascular Cognitive Impairment and Dementia (VCID)

Vascular cognitive impairment and dementia (VCID) is the most common etiology of dementia in the elderly. Both, vascular and Alzheimer’s disease, pathologies work synergistically to create neurodegeneration and cognitive impairments. The main causes of VCID include hemorrhage/microbleed (i.e., hyperhomocysteinemia), cerebral small vessel disease, multi-infarct dementia, severe hypoperfusion (i.e., bilateral common carotid artery stenosis), strategic infarct, angiopathy (i.e., cerebral angiopathy), and hereditary vasculopathy (i.e., cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). In this review, we will discuss the experimental animal models that have been developed to study these pathologies. We will discuss the limitations and strengths of these models and the important research findings that have advanced the field through the use of the models.     

代谢性疾病实验动物模型的建立与评价

代谢综合征是一组以多种代谢性疾病合并出现为临床特点的临床症候群。建立适合的MS动物模型对研究代谢性疾病的发病机理和相关药物筛选有十分重要的意义。本文就目前MS实验动物模型的建立做一综述,并对其进行评价。     

Specific Imaging of Inflammation with the 18kDa Translocator Protein Ligand DPA-714 in Animal Models of Epilepsy and Stroke

Inflammation is a pathophysiological hallmark of many diseases of the brain. Specific imaging of cells and molecules that contribute to cerebral inflammation is therefore highly desirable, both for research and in clinical application. The 18 kDa translocator protein (TSPO) has been established as a suitable target for the detection of activated microglia/macrophages. A number of novel TSPO ligands have been developed recently. Here, we evaluated the high affinity TSPO ligand DPA-714 as a marker of brain inflammation in two independent animal models. For the first time, the specificity of radiolabeled DPA-714 for activated microglia/macrophages was studied in a rat model of epilepsy (induced using Kainic acid) and in a mouse model of stroke (transient middle cerebral artery occlusion, tMCAO) using high-resolution autoradiography and immunohistochemistry. Additionally, cold-compound blocking experiments were performed and changes in blood-brain barrier (BBB) permeability were determined. Target-to-background ratios of 2 and 3 were achieved in lesioned vs. unaffected brain tissue in the epilepsy and tMCAO models, respectively. In both models, ligand uptake into the lesion corresponded well with the extent of Ox42- or Iba1-immunoreactive activated microglia/macrophages. In the epilepsy model, ligand uptake was almost completely blocked by pre-injection of DPA-714 and FEDAA1106, another high-affinity TSPO ligand. Ligand uptake was independent of the degree of BBB opening and lesion size in the stroke model. We provide further strong evidence that DPA-714 is a specific ligand to image activated microglia/macrophages in experimental models of brain inflammation.     

Exploring the Validity of Proposed Transgenic Animal Models of Attention-Deficit Hyperactivity Disorder (ADHD)

Attention-deficit/hyperactivity disorder (ADHD) is a common, behavioral, and heterogeneous neurodevelopmental condition characterized by hyperactivity, impulsivity, and inattention. Symptoms of this disorder are managed by treatment with methylphenidate, amphetamine, and/or atomoxetine. The cause of ADHD is unknown, but substantial evidence indicates that this disorder has a significant genetic component. Transgenic animals have become an essential tool in uncovering the genetic factors underlying ADHD. Although they cannot accurately reflect the human condition, they can provide insights into the disorder that cannot be obtained from human studies due to various limitations. An ideal animal model of ADHD must have face (similarity in symptoms), predictive (similarity in response to treatment or medications), and construct (similarity in etiology or underlying pathophysiological mechanism) validity. As the exact etiology of ADHD remains unclear, the construct validity of animal models of ADHD would always be limited. The proposed transgenic animal models of ADHD have substantially increased and diversified over the years. In this paper, we compiled and explored the validity of proposed transgenic animal models of ADHD. Each of the reviewed transgenic animal models has strengths and limitations. Some fulfill most of the validity criteria of an animal model of ADHD and have been extensively used, while there are others that require further validation. Nevertheless, these transgenic animal models of ADHD have provided and will continue to provide valuable insights into the genetic underpinnings of this complex disorder.     

免疫性血小板减少性紫癜动物模型建立方法与应用评价

免疫性血小板减少性紫癜(immune thrombocytopenic purpura,ITP)是血液系统自身免疫性疾病,临床以皮肤、黏膜自发性出血为主要症状。鉴于ITP发病机理目前尚不清晰,因此,探讨人类ITP发病相近或相似的动物模型有助于对ITP发病机制的认识与治疗效果的评估。但就目前状况分析,被动型与主动型的ITP造模方法虽对研究ITP发病机制与药理学研究提供了研究工具,但也存在一些尚待解决的实际问题。因此,系统回顾ITP动物模型复制方法,并给予适当评价,有助于发现、应用并完善现有的动物模型,也能更好的探索新的ITP动物模型来研究发病过程与药物治疗的效应机制。     

Evaluation of a HIV Voluntary Opt-Out Screening Program in a Singapore Hospital

Background

Early diagnosis of human immunodeficiency virus (HIV) allows for appropriately timed interventions with improved outcomes, but HIV screening among asymptomatic persons and the general population in Singapore remains low. In 2008, Singapore’s Ministry of Health implemented HIV voluntary opt-out screening (VOS) for hospitalised adults. We evaluated the outcome of VOS and surveyed reasons for its low uptake in our institution.

Methods

We assessed the outcomes of the VOS programme from January 2010 to December 2013 at National University Hospital, a 1081-bed tertiary hospital in Singapore. We also examined reasons for opting-in and opting-out using an interviewer–administered structured questionnaire in a representative sample in January 2013.

Results

107,523 patients fulfilled VOS criteria and were offered HIV screening, of which 5215 (4.9%) agreed to testing. 4850 (93.1%) of those who opted-in had an HIV test done. Three (0.06%) tested positive for HIV. 238 patients (14.2%) were surveyed regarding reasons for opting-in or out of VOS. 21 (8.8%) had opted-in. Patients who opted-in were likely to be younger, more educated and reported having more regular sexual partners. Type of housing, number of casual sexual partners, sexual orientation, intravenous drug use, condom use and previous sexually transmitted infection were not associated with deciding to opt-in/out. Patients’ most common reasons for opting-out were: belief that they were at low risk (50.2%), belief that they were too old (26.8%), cost (6.9%) and aversion to venepuncture (6.5%). The most common reason for opting-in was desire to know their HIV status (47.6%).

Conclusion

The success of an HIV-VOS program is largely determined by test uptake. Our study showed that the majority of eligible VOS patients opted-out of HIV screening. Given the considerable cost and low yield of this programme, more needs to be done to better equip patients in self-risk assessment and opting in to testing.     

Animal Models of Seizures and Epilepsy: Past,Present, and Future Role for the Discovery of Antiseizure Drugs

The identification of potential therapeutic agents for the treatment of epilepsy requires the use of seizure models. Except for some early treatments, including bromides and phenobarbital, the antiseizure activity of all clinically used drugs was, for the most part, defined by acute seizure models in rodents using the maximal electroshock and subcutaneous pentylenetetrazole seizure tests and the electrically kindled rat. Unfortunately, the clinical evidence to date would suggest that none of these models, albeit useful, are likely to identify those therapeutics that will effectively manage patients with drug resistant seizures. Over the last 30 years, a number of animal models have been developed that display varying degrees of pharmacoresistance, such as the phenytoin- or lamotrigine-resistant kindled rat, the 6-Hz mouse model of partial seizures, the intrahippocampal kainate model in mice, or rats in which spontaneous recurrent seizures develops after inducing status epilepticus by chemical or electrical stimulation. As such, these models can be used to study mechanisms of drug resistance and may provide a unique opportunity for identifying a truly novel antiseizure drug (ASD), but thus far clinical evidence for this hope is lacking. Although animal models of drug resistant seizures are now included in ASD discovery approaches such as the ETSP (epilepsy therapy screening program), it is important to note that no single model has been validated for use to identify potential compounds for as yet drug resistant seizures, but rather a battery of such models should be employed, thus enhancing the sensitivity to discover novel, highly effective ASDs. The present review describes the previous and current approaches used in the search for new ASDs and offers some insight into future directions incorporating new and emerging animal models of therapy resistance.     

The IRMM—International Measurement Evaluation Program (IMEP)

The aim of the IRMM—International Measurement Evaluation Program (IMEP) is to test out a possible realization of international measurement comparability for field laboratories through traceability of their measurements to the SI Unit for amount of substance: the mole. In IMEP-3, 10 different trace elements, B, Ca, Cd, Cu, Fe, K, Li, Pb, Rb, and Zn, were determined in a synthetic and a natural water by participating laboratories using their routine methods and graphically compared (in coded form) to certified values, established by IRMM and NIST using an isotope-specific method (Isotope Dilution Mass Spectrometry, Neutron Activation Analysis). The number of participants was 70; 64 laboratories have reported results. The results show a spread of more than 50% asymmetrically around the certified value. The Youden graphs allow evaluation of the overall performance of the laboratories in the IMEP-3 round.

     

Dynamic Network Drivers of Seizure Generation,Propagation and Termination in Human Neocortical Epilepsy

The epileptic network is characterized by pathologic, seizure-generating ‘foci’ embedded in a web of structural and functional connections. Clinically, seizure foci are considered optimal targets for surgery. However, poor surgical outcome suggests a complex relationship between foci and the surrounding network that drives seizure dynamics. We developed a novel technique to objectively track seizure states from dynamic functional networks constructed from intracranial recordings. Each dynamical state captures unique patterns of network connections that indicate synchronized and desynchronized hubs of neural populations. Our approach suggests that seizures are generated when synchronous relationships near foci work in tandem with rapidly changing desynchronous relationships from the surrounding epileptic network. As seizures progress, topographical and geometrical changes in network connectivity strengthen and tighten synchronous connectivity near foci—a mechanism that may aid seizure termination. Collectively, our observations implicate distributed cortical structures in seizure generation, propagation and termination, and may have practical significance in determining which circuits to modulate with implantable devices.     

Scale Invariance Properties of Intracerebral EEG Improve Seizure Prediction in Mesial Temporal Lobe Epilepsy

Although treatment for epilepsy is available and effective for nearly 70 percent of patients, many remain in need of new therapeutic approaches. Predicting the impending seizures in these patients could significantly enhance their quality of life if the prediction performance is clinically practical. In this study, we investigate the improvement of the performance of a seizure prediction algorithm in 17 patients with mesial temporal lobe epilepsy by means of a novel measure. Scale-free dynamics of the intracerebral EEG are quantified through robust estimates of the scaling exponents—the first cumulants—derived from a wavelet leader and bootstrap based multifractal analysis. The cumulants are investigated for the discriminability between preictal and interictal epochs. The performance of our recently published patient-specific seizure prediction algorithm is then out-of-sample tested on long-lasting data using combinations of cumulants and state similarity measures previously introduced. By using the first cumulant in combination with state similarity measures, up to 13 of 17 patients had seizures predicted above chance with clinically practical levels of sensitivity (80.5%) and specificity (25.1% of total time under warning) for prediction horizons above 25 min. These results indicate that the scale-free dynamics of the preictal state are different from those of the interictal state. Quantifiers of these dynamics may carry a predictive power that can be used to improve seizure prediction performance.     

Evaluation of TorsinA as a Target for Parkinson Disease Therapy in Mouse Models

Parkinson disease (PD) is a common and disabling disorder. No current therapy can slow or reverse disease progression. An important aspect of research in this field is target validation, a systematic approach to evaluating the likelihood that modification of a certain molecule, mechanism or biological pathway may be useful for the development of pharmacological or molecular treatments for the disease. TorsinA, a member of the AAA+ family of chaperone proteins, has been proposed as a potential target of neuroprotective therapy. TorsinA is found in Lewy bodies in human PD, and can suppress toxicity in cellular and invertebrate models of PD. Here, we evaluated the neuroprotective properties of torsinA in mouse models of PD based on intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as well as recombinant adeno associated virus (rAAV) induced overexpression of alpha-synuclein (α-syn). Using either transgenic mice with overexpression of human torsinA (hWT mice) or mice in which torsinA expression was induced using an rAAV vector, we found no evidence for protection against acute MPTP intoxication. Similarly, genetic deletion of the endogenous mouse gene for torsinA (Dyt1) using an rAAV delivered Cre recombinase did not enhance the vulnerability of dopaminergic neurons to MPTP. Overexpression of α-syn using rAAV in the mouse substantia nigra lead to a loss of TH positive neurons six months after administration, and no difference in the degree of loss was observed between transgenic animals expressing forms of torsinA and wild type controls. Collectively, we did not observe evidence for a protective effect of torsinA in the mouse models we examined. Each of these models has limitations, and there is no single model with established predictive value with respect to the human disease. Nevertheless, these data do seem to support the view that torsinA is unlikely to be successfully translated as a target of therapy for human PD.