Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

The potential of gene therapies for spinal cord injury repair:a systematic review and meta-analysis of pre-clinical studies

Currently,there is no cure for traumatic spinal co rd injury but one therapeutic approach showing promise is gene therapy.In this systematic review and meta-analysis,we aim to assess the efficacy of gene therapies in pre-clinical models of spinal cord injury and the risk of bias.In this metaanalysis,registe red at PROSPERO(Registration ID:CRD42020185008),we identified relevant controlled in vivo studies published in English by searching the PubMed,Web of Science,and Embase databases.No restrictions of the year of publication were applied and the last literature search was conducted on August 3,2020.We then conducted a random-effects meta-analysis using the restricted maximum likelihood estimator.A total of 71 studies met our inclusion crite ria and were included in the systematic review.Our results showed that overall,gene therapies were associated with improvements in locomotor score(standardized mean difference[SMD]:2.07,95%confidence interval[CI]:1.68-2.47,Tau^(2)=2.13,I^(2)=83.6%)and axonal regrowth(SMD:2.78,95%CI:1.92-3.65,Tau^(2)=4.13,I^(2)=85.5%).There was significant asymmetry in the funnel plots of both outcome measures indicating the presence of publication bias.We used a modified CAMARADES(Collaborative Approach to M eta-Analysis and Review of Animal Data in Experimental Studies)checklist to assess the risk of bias,finding that the median score was 4(IQR:3-5).In particula r,reports of allocation concealment and sample size calculations were lacking.In conclusion,gene therapies are showing promise as therapies for spinal co rd injury repair,but there is no consensus on which gene or genes should be targeted.

Astrocytes dynamically regulate the blood-brain barrier in the healthy brain

The blood-brain barrier(BBB)(discovered and defined by Max Lewandowsky and Lina Stern,and not,as it is universally,and yet erroneously believed,by Paul Ehrlich(Verkhratsky and Pivoriunas,2023))that separates the nervous system from the circulation is evolutionarily conserved from arthropods to man.The primeval BBB of the invertebrates and some early vertebrates was made solely by glial cells and secured(in invertebrates)by septate junctions.

Structural changes in the obese brain

Obesity represents a major global health concern,with widespread growing prevalence and severe consequences for the quality of life and life expectancy of affected individuals and for the economic burden of healthcare systems(Prospective Studies Collaboration,2009).Obesity is a well-established risk factor for a variety of conditions including metabolic,vascula r,and heart diseases,and seve ral types of cancer(Prospective Studies Collaboration,2009).

Astrocyte chloride,excitatory-inhibitory balance and epilepsy

Excitation and inhibition are at the core of brain function and malfunction.To sustain the activity of neuronal networks over time and space,glutamatergic excitation is balanced by GABAergic inhibition.The equipoise of excitation and inhibition,known as the excitation/inhibition(E/I)balance,is crucial for proper brain function.The E/I balance is highly dynamic and shifts across different brain states:wakefulness primarily augments excitatory activity,while sleep promotes a decrease in excitation and an increase in inhibition(Bridi et al.,2020).Neuronal activity during various brain states is primarily regulated by neurotransmitters(Schiemann et al.,2015),alongside non-synaptic mechanisms that operate on a slower timescale.The non-synaptic mechanisms are many,with the ionic composition of the extracellular space playing a significant role;altering extracellular ion concentrations affects sleep,arousal,electroencephalogram patterns,and behavioral states(Ding et al.,2016).

Magnesium acethyltaurate as a potential agent for retinal and optic nerve protection in glaucoma

Glaucoma is the second leading cause of irreversible vision impairment affecting more than 70 million people worldwide with approximately 10%suffering from glaucoma-related bilateral blind（Quigley and Broman,2006）.It is a multi-factorial disease that is characterized by optic nerve damage and visual field loss.Progressive loss of retinal ganglion cells（RGCs）resulting in visual field deficits is the hallmark of glaucoma.

Comparison of ELISA and HPLC-MS methods for the determination of exenatide in biological and biotechnology-based formulation matrices

The development of biotechnology-based active pharmaceutical ingredients, such as GLP-1 analogs, brought changes in type 2 diabetes treatment options. For better therapeutic efficiency, these active pharmaceutical ingredients require appropriate administration, without the development of adverse effects or toxicity. Therefore, it is required to develop several quantification methods for GLP-1 analogs products, in order to achieve the therapeutic goals, among which ELISA and HPLC arise. These methods are developed, optimized and validated in order to determine GLP-1 analogs, not only in final formulation of the active pharmaceutical ingredient, but also during preclinical and clinical trials assessment. This review highlights the role of ELISA and HPLC methods that have been used during the assessment for GLP-1 analogs, especially for exenatide.

实验性变应性鼻炎鼻粘膜神经生长因子的测定

目的 探讨神经生长因子 (nervegrowthfactor,NGF)在变应性鼻炎发病中的作用。方法建立SD大鼠变应性鼻炎的模型 ,利用酶联免疫吸附测定 (enzyme linkedimmunosorbentassay,ELISA)法测定实验性变应性鼻炎鼻粘膜组织中的神经生长因子的含量。结果 实验性变应性鼻炎鼻粘膜的神经生长因子含量 ( x±s,下同 ;n =9)为 (6 .6 5± 0 .42 )ng/g湿重 ,对照组 (n =6 )为 (3.5 1± 0 .2 9)ng/g湿重 ,二者相比差异有显著性 (P <0 .0 0 1)。结论 神经生长因子可能通过调节神经肽的表达和释放和/或者通过活化免疫细胞 (如肥大细胞 )

PrP^C-related signal transduction is influenced by copper, membrane integrity and the alpha cleavage site

The copper-binding, membrane-anchored, cellular prion protein （PrP~） has two constitutive cleavage sites producing distinct N- and C-terminal fragments （N1/C1 and N2/C2）. Using RK13 cells expressing either human PrPc, mouse PrPc or mouse PrP^C carrying the 3F4 epitope, this study explored the influence of the PrP^C primary sequence on endoproteolytic cleavage and one putative PrPc function, MAP kinase signal transduction, in response to exogenous copper with or without a perturbed membrane environment. PrPc primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat- ed kinase 1/2 （ERK1/2） phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrP^C showed increased N1/C1 cleavage in response to copper alone, accompanied by specific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrP^C-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPc harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPc pri- mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence appears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of PrP^C-related signal transduction in response to exogenous stimuli.

Colony stimulating factor 1:friend or foe of neurons?

Colony stimulating factor 1 receptor(CSF1R) is a tyrosine kinase receptor primarily expressed on microglia and a small subpopulation of neurons in the central nervous system (CNS),which directly controls the homeostasis,activation,and proliferation of microglia.Its ligands include CSF1 and interleukin-34 (IL-34).

Astrocytes in human central nervous system diseases: a frontier for new therapies

Astroglia are a broad class of neural parenchymal cells primarily dedicated to homoeostasis and defence of the central nervous system(CNS).Astroglia contribute to the pathophysiology of all neurological and neuropsychiatric disorders in ways that can be either beneficial or detrimental to disorder outcome.Pathophysiological changes in astroglia can be primary or secondary and can result in gain or loss of functions.Astroglia respond to external,non-cell autonomous signals associated with any form of CNS pathology by undergoing complex and variable changes in their structure,molecular expression,and function.In addition,internally driven,cell autonomous changes of astroglial innate properties can lead to CNS pathologies.Astroglial pathophysiology is complex,with different pathophysiological cell states and cell phenotypes that are context-specific and vary with disorder,disorder-stage,comorbidities,age,and sex.Here,we classify astroglial pathophysiology into(i)reactive astrogliosis,(ii)astroglial atrophy with loss of function,(iii)astroglial degeneration and death,and(iv)astrocytopathies characterised by aberrant forms that drive disease.We review astroglial pathophysiology across the spectrum of human CNS diseases and disorders,including neurotrauma,stroke,neuroinfection,autoimmune attack and epilepsy,as well as neurodevelopmental,neurodegenerative,metabolic and neuropsychiatric disorders.Characterising cellular and molecular mechanisms of astroglial pathophysiology represents a new frontier to identify novel therapeutic strategies.

Copper interactions with DNA of chromatin and its role in neurodegenerative disorders

In this study, we have demonstrated the conformational changes to DNA induced by abnormal interactions of copper using circular dichroism, in combination with UV-absorbance and fluorescence spectroscopy. Results confirm that binding of copper to bases of DNA in chromatin is concentration dependent. Binding efficiency of Cu2~ ions to DNA is increased in proportion to the degree of unwinding of the double helix induced by denaturation. Altered B-DNA conformation will alter the integrity of DNA which may affect the normal process of DNA replication and transcription. Copper induced DNA damage in the brain may cause neurotoxicity and the neuronal cell death and is implicated in Alzheimer＇s disease and other neurological disorders.

New models to study vulvodynia: hyperinnervation and nociceptor sensitization in the female genital tract

Vulvodynia is a prevalent form of chronic pain, most com- monly affecting the vaginal vestibule （vestibulodynia） （Pukall et al., 2016）. Women with vulvodynia describe intense pain in response to light touch of the affected region, such that sexual function and other activities can be severely limited. Medical costs associated with vulvodynia are high, exceeding $21 billion annually in the United States （Xie et al., 2012）. The high level of direct medical costs has been linked to high treatment failure rates. Many women with the disorder consult multiple practitioners and undergo multiple courses of treatment with limited benefit.

Pharmacological interventions targeting nuclear factor-kappa B signaling in multiple sclerosis

Multiple sclerosis(MS)is an inflammatory neurodegenerative disease of the central nervous system(CNS).Pathological characteristics of the disease include activation of CNS-intrinsic immune cells,such as microglia and astrocytes,and loss of neuronal connections,myelin and blood-brain barrier(BBB)integrity as well as peripheral immune cell infiltration into the brain.MS has long been considered a predominantly immunological disease,which has led to the development of essentially only immune-directed medications.Within this traditional“outside-in”MS hypothesis,a dysregulation of the peripheral immune system causes immune cell infiltration into the CNS,leading to autoreactivity against myelin sheath components and secondary BBB dysfunction.However,recent findings indicate that overactivation of microglia and astrocytes represents an important first step in MS pathology,as appears to be the case for other neurodegenerative diseases such as Alzheimer’s disease(AD)and Parkinson’s disease(PD).

Astrocyte-derived extracellular vesicles mediate intercellular communications of the neurogliovascular unit

The cortical grey matter of mammals has a specific cyto-architecture defined by the process of "tiling" in which protoplasmic astrocytes parcellate the nervous tissue into spatially segregated territorial domains.

Heuristics in Language Comprehension

We used a sentence-picture matching task to demonstrate that heuristics can influence language comprehension. Interpretation of quantifier scope ambiguous sentences such as Every kid climbed?a tree was investigated. Such sentences are ambiguous with respect to the number of trees inferred;either several trees were climbed or just one. The availability of the NOUN VERB NOUN (N-V-N) heuristic, e.g., KID CLIMB TREE, should contribute to the interpretation of how many trees were climbed. Specifically, we hypothesized that number choices for these stimuli would be predicted by choices previously made to corresponding (full) sentences. 45 participants were instructed to treat N-V-N triplets such as KID CLIMB TREE as telegrams and select a picture, regarding the quantity (“several” vs. “one”) associated with tree. Results confirmed that plural responses to quantifier scope ambiguous sentences significantly predict increased plural judgments in the picture-matching task. This result provides empirical evidence that the N-V-N heuristic, via conceptual event knowledge, can influence sentence interpretation. Furthermore, event knowledge must include the quantity of participants in the event (especially in terms of “several” vs. “one”). These findings are consistent with our model of language comprehension functioning as “Heuristic first, algorithmic second.” Furthermore, results are consistent with judgment and decision making in other cognitive domains.

Synergistic Suppression of Cortical Spreading Depression under NR2A and NR2B Inhibition

Cortical spreading depression (CSD) is a transient synaptic excitation, followed by depression, which can propagate slowly across cortex, subcortex and retina. CSD is implicated in migraine with aura and may lead to migraine pain. CSD can be suppressed by inhibition of both NR2A and NR2B containing N-methyl-D-aspartic acid (NMDA) receptors. However, whether there is a synergistic effect of co-inhibition of NR2A and NR2B containing receptors on CSD remains unknown. In this study, an efficient in vitro model of migraine with intrinsic optical imaging approach was applied to address this role of co-activation of both NR2A and NR2B in CSD. The results showed that co-application of NVP-AAM077, a drug selectively targeting for NR2A and Ro 25-6981, a drug selectively targeting for NR2B containing receptors, showed synergistic inhibitory effects on propagation rate and the magnitude of CSD in a concentration dependent manner in chick retina. Inhibition of CSD propagation was also observed by a clinical acceptable drug that antagonizes both NR2A and NR2B containing receptors, memantine, at 10 μM. These data suggest that combinational use of antagonists selectively targeting NR2A and NR2B containing receptors could be a useful strategy for preventative treatment of migraine with improved efficacy with potential alleviated side effects.

Neural correlates of focused attention in patients with mild Alzheimer’s disease

Alzheimer’s Disease (AD) is characterized by an early and significant memory impairment, and progresses to affect other cognitive domains. Impairments in Focused Attention (FA) have been observed in patients diagnosed with mild AD. A functional magnetic resonance imaging (fMRI) Stroop paradigm with verbal responses was used to investigate the neural correlates of FA in AD patients. Twenty-one patients diagnosed with mild AD performed a verbal Stroop—fMRI paradigm. Colour words were printed in an incongruent ink colour. Series 1 consisted of four blocks “Read the word” followed by four blocks “Say the colour of the ink”;Series 2 alternated between the two conditions. Functional data were analyzed using SPM5 to detect anatomical areas with significant signal intensity differences between the conditions. Within-group analyses of the colour minus word contrast yielded significant activation in the following left hemisphere regions: precentral gyrus, inferior frontal gyrus, fusiform gyrus and supplementary motor area (p < 0.05, uncorrected). Relative to cognitively normal older adults who underwent the same experimental task, Stroop performance was significantly worse in AD patients. The fMRI task yielded similar activated brain regions between the two groups. The use of verbal responses in this novel fMRI Stroop task avoids the confusion and memorizing of button locations seen with the manual response modality, allowing the neural correlates of FA to be investigated in AD patients.

Use of complementary and alternative medicines in people with depression and central obesity: Findings from a Tai Chi and Qigong study

Objective:Depression is a global and growing health issue.Complementary and alternative medicines (CAM) use is common among people with depression.We examined the baseline use of CAM (products,not services) in centrally obese adults with depression,a specific and increasingly important population who were part of a trial of Tai Chi and Qigong for depression.Methods:Self-reported use of CAM products (four major groups:vitamins,herbal medicines,mineral supplements and purified chemicals as nutritional supplements) together with demographic,lifestyle and health status characteristics were collected using validated questions within a written survey.We used descriptive statistics to understand patterns of use.Results:Two hundred and sixty patients participated in the assessment.Half of the patients stated they were using CAM,on average reporting 2.3 CAM per patient.Women were more likely than men,and older people (>60 years) more likely than younger people (<40 years) to use any CAM products.Patients reported using CAM mostly for general health and alleviating aches and pains.CAM use was not associated with health state,use of a prescription antidepressant medicine,nor (self-reported) comorbidities such as arthritis,diabetes,hypertension or alcohol or tobacco use.Conclusions:CAM products are commonly used in Australians with moderate depression,particularly in female or older patients.The range and number of CAM in this group with common comorbidity may present additional unknown risks due to potential interactions with other prescribed medications with a myriad of biological and pharmacological effects.The importance of establishing whether depressed patients are taking CAM,the particular CAM and whether the CAM interacts with each other,other drugs used for depression or the biological process of the depression itself are factors to be considered at each assessment.

运动神经元在脊髓损伤后肌痉挛发生中的作用

Motoneurons in the spinal cord have intrinsic voltage dependent persistent in ward currents (PICs; e.g. persistent calcium currents) that amplify synaptic inp uts by three to five fold in addition to providing a sustained excitatory driv e that allows motoneurons to fire repetitively following a brief synaptic excita tion. In this study, we examined whether prolonged involuntary muscle spasms in subjects with long term injury to the spinal cord are mediated by the activatio n of PICs in the motoneuron. To examine this in the human, we used a paired moto r unit analysis technique where the firing frequency of one motor unit of the pa ir (control unit) was used to estimate the synaptic drive to the motoneuron pool , including the drive to a second higher threshold motor unit of the pair (test unit). The degree to which a motoneuron PIC helped to sustain the discharge of a test motor unit (self sustained firing) was determined from the reduction in control unit firing at de recruitment (△F) compared with recruitment of the te st unit. This △F value corresponds to the reduction in synaptic drive needed to counteract the intrinsic PIC and, thus, was used an indirect measure of this cu rrent. In the nine motor unit pairs studied, the average estimated synaptic driv e, or control unit firing rate, required to recruit a test motor unit at the ons et of a muscle spasm was significantly higher (by 43%) than the estimated synap tic drive during derecruitment at the end of a muscle spasm. This indicated that a motoneuron PIC, and associated self sustained firing, facilitated the firing of the test units during the prolonged muscle spasms. In addition, in all subje cts tested (seven out of seven), we observed that following a muscle spasm or vo luntary contraction, spontaneous and self sustained firing of motor units could continue for many seconds, even minutes, at very low discharge rates (average 5.2 ±.1.6 Hz) with extremely low spike to spike variability (coefficient of v ariation = 5.4 ±.1.6%). Moreover, increases in synaptic drive (noise) to the s pontaneously firing units with voluntary muscle contractions or muscle spasms in creased both the mean firing rate of the motor units in addition to their firing variability. This suggests that the slow spontaneous firing commonly observed i n chronic spinal injury likely occurs without appreciable synaptic noise and is likely driven to a substantial degree by PICs intrinsic to the motoneuron becaus e it is self sustained and very regular.