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string | predicted_class
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|---|---|---|
HIF-1 has been shown to regulate certain JmjC enzymes previously. As such, KDM3A, KDM4B, KDM4C, KDM5C, and KDM6B have all been shown to be HIF targets (reviewed in ). However, despite some reports indicating that KDM2A and KDM2B mRNA levels increase in response to hypoxia , there was no information as to whether these where HIF-dependent genes.
|
review
| 99.9 |
KDM2B has been previously shown to be nuclear factor (NF)-κB dependent, and be negatively regulated by TRAIL treatment in several cancer cell lines . While we have previously shown that hypoxia activated NF-κB to modulate its targets , we have not investigated if hypoxia-induced KDM2B mRNA is NF-κB dependent. We did find that depletion of HIF-1 significantly decreased KDM2B mRNA in hypoxia. Moreover, recently, we demonstrated that HIF-1α acts as a repressor of NF-κB in cells, controlling its activity both in normoxia and hypoxia , thus suggesting that under these conditions, NF-κB does not control KDM2B expression. Intriguingly, hypoxia exposure results in reduced protein levels of KDM2B in U2OS, in a HIF-1-dependent manner, suggesting that HIF-1-mediated effects on KDM2B protein are not direct and are post-transcriptional. How KDM2B protein is regulated in U2OS cells is currently unknown and would thus require additional work.
|
study
| 100.0 |
Our data clearly show that KDM2A is a novel HIF-1α-dependent target. Transcript and protein levels increase in hypoxia in a HIF-1α-dependent manner in both cell lines tested. In addition, we could demonstrate that HIF-1α and HIF-1β are present at the KDM2A promoter and that this is required for recruitment of RNA polymerase II to this promoter. A study analysing RNA polymerase II behaviour in hypoxia has revealed that some promoters already possess RNA polymerase loaded but it was non-processive . Hypoxia was shown to release RNA polymerase from these promoters and thus induce transcription of these targets . However, the authors did mention exceptions such as ADM, for example, where hypoxia induced RNA polymerase II loading . Furthermore, given that the resolution of the ChIP-qPCR is around 400 bp, our results could also be consistent with a HIF-dependent RNA polymerase II release as proposed by the abovementioned study.
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study
| 100.0 |
KDM2A has been reported to act as an oncogene in different types of cancer. As such, it has been shown to promote tumourigenesis in lung and gastric cancers, and more recently an oncogenic isoform of KDM2A has been identified in breast cancer . However, KDM2A has also been shown to repress rRNA transcription, a process required for cell proliferation ; also, when phosphorylated by ataxia-telangiectasia mutated (ATM), KDM2A helps in DNA damage repair . Interestingly, HIF-1α also has dual roles in cancer, being important for tumourigenesis in cancer such as colon and breast , but acting more as a tumour suppressor in cancers such as renal clear cell cancer .
|
review
| 99.8 |
Given that KDM2 enzymes require oxygen for their histone demethylase function, it would be tempting to speculate that, despite the increase in expression, their demethylase activity would still be low. However, their other protein domains would still be functional, and as such their role as ubiquitin ligases might be increased in hypoxia. However, currently very few targets are known for these proteins and, as such, further work directed at these questions is therefore need. It would thus be interesting to analyse which proteins KDM2 family members interact with and modify, and what the functional consequences are for the cell, when KDM2 levels increase or decrease. Given that the KDM2 family targets important histone methylation marks such histone H3 lysine 4 trimethylation (H3K4me3) and H3K36me/me2 , regulation of KDM2 abundance will impinge into the levels of these histone marks. This suggests that prolonged hypoxia could therefore change chromatin structure via the mechanism described here, where HIF-1 induces KDM2 protein changes. Analysis of histone marks in cells exposed to different periods of hypoxia would allow to determine how the action of KDM2 proteins is controlled under such stress conditions.
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study
| 100.0 |
Age-related macular degeneration (AMD) is the leading cause of vision loss in the developed world.1 There are two main forms of AMD. The first is non-neovascular (dry) AMD which accounts for the majority of AMD cases and results from the deposition of drusen (small yellow or white deposits) underneath the retina that eventually leads to the slow degeneration of retinal cells resulting in blindness. Neovascular (wet) AMD (nvAMD) accounts for the remaining cases of AMD and results from the development of new blood vessels deep to the retina which leak or bleed resulting in symptoms of new distortion or vision loss. nvAMD results in irreversible blindness if left untreated and accounts for 90% of the cases of blind registration resulting from AMD.2 The main risk factors associated with AMD are age and smoking.3 Cases of blindness resulting from AMD are predicted to increase together with an ageing population.4
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review
| 99.9 |
An effective treatment is currently available to preserve vision in nvAMD in the form of intravitreal therapy (IVT) with antivascular endothelial growth factor agents.5–8 These drugs have been shown to be effective in maintaining long-term vision in the majority of patients affected by nvAMD.9 Delay in instituting IVT treatment in new cases of nvAMD has been shown to be one of the most important factors negatively impacting final visual outcome.10 11 Consequently, the early diagnosis and treatment is crucial to improving visual outcomes in AMD and to reduce the social and economic burden of blindness resulting from the disease.12 13
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review
| 99.9 |
Delays from symptom onset to treatment can be experienced at different stages of the patient care pathway for new onset nvAMD. These include: (1) time of first symptom onset to presentation at primary care practitioner, (2) time from primary care referral to presentation at ophthalmic clinic and (3) time from ophthalmic clinic to first IVT treatment (figure 1).14 These early stages of the care pathway also represent the periods during which lesions may be most active and amenable to the benefits of therapy.15
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other
| 99.3 |
There have been many published reports investigating intrahospital factors such as the time from first ophthalmic clinic visit to first IVT treatment.10 11 16 However, there is a scarcity of literature reporting the extrahospital factors such as the time from symptom onset to presentation at ophthalmic clinic. In addition, despite its significance in causing blindness, limited research has been performed to investigate AMD awareness. An exploration of patient’s awareness and knowledge of disease has been demonstrated in other chronic diseases such as stroke and cancer,17 18 with increased awareness associated with improved patient outcomes.19 20
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review
| 99.7 |
The primary objectives of this study were twofold: first, to assess the time between the different stages of the nvAMD care pathway in patients treated in Southeast Scotland and second, to evaluate patients’ awareness of AMD, its risk factors and treatment options.
|
study
| 99.94 |
Case notes of consecutive patients diagnosed and treated with IVT for nvAMD in NHS Lothian since September 2013 were identified using a treatment clinic register. A 2013 cut-off point was chosen to reflect the updated guidelines on AMD by the Royal College of Ophthalmologists (RCOphth) which were published at the time.14 The guidelines recommended that all patients with suspected AMD should be seen by a retinal specialist within 1 week of referral and that treatment should commence within 1 week of first ophthalmic appointment (figure 1).
|
study
| 99.8 |
In this study, the main outcome measures were: (1) time from symptom onset to first presentation at primary care (ie, duration of visual symptoms before initial presentation), (2) time from primary care referral to ophthalmic clinic appointment and (3) time from ophthalmic clinic appointment to first IVT treatment. The main exclusion criteria were case notes with incomplete data and the coexistence of ocular comorbidities that gave rise to choroidal neovascularisation. This study was approved as part of a wider service evaluation which was accepted following review by the NHS Lothian Ophthalmology Quality Improvement Team on 8 October 2015.
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study
| 100.0 |
In order to investigate patients’ awareness of AMD, a cluster random sample of patients visiting ophthalmic clinics for non-AMD disease in NHS Lothian was surveyed using a 12-item questionnaire (see online supplementary file 1). The sample size required for the study was calculated using a power calculation (see online supplementary file 2). Questions were adapted from a previously validated questionnaire21 and served to ascertain each patient’s knowledge of AMD and its risk factors. Patients were asked for their demographic details, including age, sex, education and postal code of residence. Socioeconomic deprivation scores (social class) were calculated for all patients from postal code data at the time of interview using the Scottish Index of Multiple Deprivation (SIMD).22 The SIMD combines weighted data on seven domains (income, employment, education, housing, health, crime and geographical access) and is officially sanctioned by the Scottish Government as a measure of multiple deprivation.23 24
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study
| 100.0 |
The first part of the questionnaire explored patients’ familiarity with AMD and its risk factors. The second part enquired about patients’ smoking status and their awareness of available treatments for AMD. Surveys were distributed and collected by the same researcher, who remained nearby to answer any questions about instructions. No additional assistance was provided. The survey was performed from 18 November 2015 to 31 November 2015 and data were analysed using Pearson χ2 tests except for education and social class where χ2 tests for trend were performed. Data analysis was done using IBM SPSS Statistics for Windows, V.23 (IBM, Armonk, New York, USA).
|
study
| 99.94 |
A total of 315 case notes were identified; 120 of the 315 were excluded after application of the exclusion criteria (see online supplementary data for the demographics and breakdown of excluded cases), leaving 195 case notes for analysis. One hundred and twenty (61.5%) patients were women, with a mean age of 78 years. Nearly all patients (187; 95.9%) presented with nvAMD affecting the first eye. The overall mean time from symptom onset to presentation was 54.2 (95% CI ±13) days. As for referrals to ophthalmology, 118 (60.5%) of these were direct from optometrists, 5 (2.6%) were direct from general practitioners (GPs) and 52 (26.7%) were made by optometrists via GPs. The remaining referrals were from other hospitals, other ophthalmology clinics and screening programmes.
|
study
| 99.94 |
The mean time from referral to ophthalmic clinic appointment was 28.2 days (95% CI ±4.0 days). There was a significant additional mean delay of 7.5 (P<0.05) (95% CI ±1.6) days when patients were referred from their optometrist via their GP. During clinic appointments, fundus fluorescein angiogram was performed in approximately one-third of patients (66/195). The mean time from clinic to first IVT treatment was 31.5 (95% CI ±3.6) days (figure 2).
|
study
| 99.94 |
The delay from symptom onset to first injection resulted from both intrahospital and extrahospital factors. We have already identified that when optometrists referred via the GPs instead of directly to the hospital eye service this resulted in a significant increased delay. However, even this delay is overshadowed by the mean delay from symptom onset to presentation at primary care service. In order to better understand patient factors that may have resulted in this delay in presentation we performed a questionnaire survey on patients with unrelated disease in the eye service. A total of 142 patients were approached in non-AMD ophthalmic clinics. These clinics included glaucoma, ocular motility and general outpatient clinics. One hundred and forty patients agreed to participate. Two refused because of unwillingness and inability to understand the purpose of the questionnaire due to deafness respectively.
|
study
| 100.0 |
The cohort included 61 (43.6%) men and 79 (56.4%) women with a median age of 73 (range: 17–93) years, comprising all social classes. The education level of patients ranged from primary education to university degree. Details of the demographic data are given in table 1.
|
study
| 99.94 |
Of the 140 respondents, 87 (62.1%) reported being ‘aware’ of AMD. Fourteen (10%) had previously been diagnosed with AMD. Out of these 14 patients (71.4%), 10 were able to provide a correct description of the symptoms of AMD. For those patients without a prior diagnosis, only 47/126 (37.3%) were able to correctly report the symptoms of AMD. There was a significant difference when comparing the responses of those who had a previous diagnosis of AMD to those without AMD (P=0.013). Overall, female respondents were more likely than male respondents to report awareness of AMD (P=0.015) (table 2). Increased awareness of AMD was also seen with higher levels of education (P=0.001).
|
study
| 99.94 |
The top risk factor for AMD correctly considered by patients was age (127/140–90.7%). The other risk factors identified included smoking in 82 (58.6%), unprotected UV exposure in 62 (44.3%), genetic predisposition in 62 (44.3%), vitamin deficiency in 54 (38.6%) and gender in 15 (10.7%).
|
study
| 100.0 |
Eighty-seven (62.1%) of patients thought that AMD was a treatable condition. However, only 20/87 (23%) were able to provide correct information on the available treatments (ie, eye injections and laser therapy). The majority of patients (91/140, 65%) considered opticians to be their first port of call if they had vision problems. Other healthcare professionals cited as first port of call included GPs in 28 (20%) and ophthalmologists in 21 (15%).
|
study
| 65.06 |
The RCOphth has recently updated its guidance on suggested waiting times for IVT treatment in nvAMD in the hospital setting. It recommends that all patients should be seen by a retinal specialist within 1 week of primary care referral, and should begin treatment within 1 week following this.14 The new guidelines place increased importance on correct diagnosis and urgent referral from primary care and place increasing emphasis on hospital eye services to provide capacity for urgent new AMD cases in addition to the treatment of existing patients with nvAMD. However, this study finds that there are significant delays at each step of the nvAMD care pathway in south-east Scotland; both the waiting times from: (1) primary care referral to ophthalmic clinic and (2) initial ophthalmic assessment to treatment are about four times as long as the recommended gold standard. In addition, there is a further 1-week delay on average when indirect referrals are made by optometrists via GP. Similar findings have also been reported in previous studies which have demonstrated similar, if not longer, delays for intrahospital pathways (ie, from initial ophthalmic assessment to treatment).10 11 16
|
study
| 99.9 |
Delays from intrahospital pathways may be attributed to the inherent diagnostic and referral pathways within different healthcare systems. In Southeast Scotland, a new IT scheme linking community optometrists and eye clinics within hospitals across all of Scotland was introduced in 2010 following a successful pilot scheme in NHS Fife which allowed optometrists to make direct electronic referrals to ophthalmologists.25 However, the system has yet to be fully integrated into all units. Our study has highlighted that there is still much room for improvement for both the primary care referral system, and also within the acute referral clinics themselves. The current electronic system still relies on a manual, ad hoc system for making referrals. An important step forward would be to develop a semiautomated referral system so that eye care providers can track patient referrals, obtain data on patient leakages and receive automatic notifications when there is lack of follow-up.
|
other
| 98.9 |
To our knowledge, this is the first study to evaluate the time from symptom onset to presentation at clinic (extrahospital pathway) for patients with nvAMD in the UK. There are, however, several limitations to the study. First, assessment of presentation delay might be difficult due to the retrospective nature of evaluation of symptom onset by patients. Second, the perception of symptoms is also highly subjective, often depending on factors such as existing cognitive function, ocular dominance of the affected eye and baseline visual acuity of the unaffected eye. Nevertheless, it is noteworthy that this time interval often varies widely between patients and is prolonged in most cases. Therefore, although less accurate than formal angiographic diagnosis, we thought it is important to investigate this time interval as it would be accessible to intervention.
|
study
| 99.94 |
Our findings demonstrate that presentation delay represents a major source of delay and accounts for the greatest proportion of the delay in the nvAMD care pathway in Southeast Scotland. This represents an important target for improvement to reduce vision loss resulting from delay in the nvAMD care pathway.26 This delay is likely to be complex and multifactorial, involving patients, eye care providers and healthcare systems. Barriers to early presentation might include a lack of awareness of AMD among patients, self-examination by patients and screening of the disease by non-retina specialists. This can be further compounded by issues such as transport difficulties, age-related infirmity and a mismatch between patient expectations on speed of referral and recommended guidelines.27 Further studies are warranted into the reasons underlying our findings in both primary care and hospital eye service environments in order that appropriate measures are taken to identify patients early and build service capacity accordingly.
|
study
| 100.0 |
At present, the diagnosis of new nvAMD, especially for the first affected eye, still very much relies on self-recognition of visual symptoms by patient themselves. This is, however, problematic as those affected in only one eye tend not to be aware of the visual change and may therefore remain ‘asymptomatic’ for a considerable length of time.28 Indeed, this seemed to be case in our study in which nearly all patients presented with nvAMD affecting the first eye.
|
study
| 99.9 |
There is evidence to show that the best-corrected visual acuity at the time of diagnosis of nvAMD is worse for the first affected eye when compared with that of the second eye.29 In addition, previous studies have shown that the visual prognosis of the first affected eye following 1 year of treatment is usually worse compared with that of the second affected eye in nvAMD.30 31 These better outcomes of the second affected eye are most likely due to increased awareness and more frequent monitoring of the second eye as part of a systematic bilateral follow-up examination for the first affected eye. These factors would seemingly translate into a shorter delay in presentation for the second affected eye, but it should be noted that this association was not explored in our study and remains to be investigated. Nonetheless, the considerable delay in presentation for the first affected eye demonstrated in our study highlights the importance of early detection and treatment.
|
study
| 99.94 |
From the patient’s perspective, the delay in symptom recognition can be addressed to a certain extent by self-examination. Patients, especially those with an increased risk of developing nvAMD, should be educated and made aware of symptoms such as new visual distortion and sudden reduction in vision. This can be achieved by encouraging patients to use suitable spaced self-tests of vision which examine one eye at a time to prevent compensation from the good eye. The standard Amsler test has long been recommended as the standard self-monitoring test, but there has been increasing reservation about its utility as a diagnostic tool due to its insufficient reliability and variable sensitivity.32 33 The advent of more innovative, cost-saving technologies may circumvent these issues and make implementation of self-examination on a wider public scale more feasible in the near future.34 35
|
review
| 99.4 |
In this study, we chose to investigate patients’ awareness of AMD because it is clear that a lack of disease awareness is a common factor for delayed presentation in other eye conditions such as glaucoma, retinal detachment and central retinal artery occlusion.36 37 The only previous study to investigate AMD awareness in the UK population showed a low awareness (16%).38 Our study adds to the existing literature by demonstrating that public awareness of AMD is still limited. Our survey shows that awareness of AMD is unacceptably low (37%), especially considering that this condition is the leading cause of blindness in developed countries.1 The low awareness of AMD is also consistent with the low levels of awareness of AMD in other countries including Australia, Hong Kong, Singapore, Nepal, Bangladesh, China and the USA (range between 5% and 50.5%).21 38–44 It is likely that our findings underestimate the true scale of lack of awareness among the general population because we sampled ophthalmic patients who, by virtue of being surveyed in an eye hospital, are presumably somewhat more attuned to common eye diseases. Our survey also highlights a low awareness of risk factors of AMD (other than age). However, this assessment could be limited by the lack of plausible distractors in the corresponding question which might have increased the respondent’s chances of getting a correct answer(s), hence again underestimating the true scale of lack of awareness.
|
study
| 99.94 |
These findings are important given the severity of the consequences of delayed presentation in AMD and the ready availability of an effective treatment to prevent visual loss. We identified AMD-naive male patients and those with lower education levels to have a particularly low awareness of warning symptoms of AMD, suggesting the need for targeted intervention for these subgroups. As increased awareness can lead patients to seek appropriate medical care, improving awareness would logically lead to better visual prognoses for patients.
|
study
| 99.9 |
There is currently still a need for a unified national awareness campaign on AMD in the UK. A recent report by the Royal National Institute of Blind People highlighted that most initiatives at improving AMD awareness in the UK still operate at a local level.27 Even then, these efforts often comprised of educational talks targeted at existing patients, rather than raising public awareness. The need for a national campaign has also been recognised by the Macular Society which has made increasing AMD awareness one of the main objectives of its 5-year national strategy.45
|
other
| 99.9 |
Although some progress has been made since,46 there is still room for improvement. Current awareness interventions need to be further optimised for a sustained impact. A promising step would be the adoption of the multilayered approach as adopted by other developed countries.47 This approach saw the use of a campaign which included a diverse range of activities such as promoting education programmes for patients and primary care, running a national advertising campaign and providing free mobile screening. The end of this focused campaign saw a dramatic increase in AMD awareness and the number of the population requesting fundus examination for symptoms of AMD.47 The implementation of a similar public health strategy in the UK may achieve similar desirable effects, but further research is needed to evaluate the effectiveness of this approach in the UK population. Another important gap highlighted by our study is the underappreciated link between smoking and AMD. This represents a potent novel health promotional tool and awareness could be increased by incorporating information in existing campaigns with other smoking-related diseases.48 49
|
study
| 99.7 |
Lack of awareness and knowledge of correct referral among non-ophthalmologists is also problematic. This may account for the delay in referral demonstrated in our study. A recent national survey revealed that 32% of GPs felt ‘deskilled’ in diagnosing common eye conditions.50 The same survey also showed that 38% of GPs felt that eyes are the most difficult part of the body to diagnose. Achieving a better alignment of ophthalmic knowledge between healthcare organisations and professionals will help improve understanding and management of common ophthalmic disorders for those in the front line of eye care.
|
study
| 99.9 |
There is significant delay at every step of the care pathway for patients with nvAMD in Southeast Scotland. We also show that awareness and knowledge of AMD are suboptimal. This lack of AMD awareness could account for the long presentation delay of AMD to primary care. This suggests that efforts to educate the public regarding AMD may lead to earlier presentation and hence improved visual outcomes in patients.
|
study
| 86.56 |
World Health Organization has identified mood disorders as a major cause of mortality, morbidity and disability worldwide [1, 2]. Social defeat is a critical phenotype of mood disorders, including severe depression, anxiety, bipolar disorder, autism and schizophrenia. Also social defeat is manifested by symptoms such as social fear, social avoidance and social withdrawal [3–5]. Overcoming social defeat is the primary determinant of the efficacy of drugs and therapeutic methods on mental disorders. However, the etiology and mechanisms of social defeat remain poorly understood.
|
review
| 99.9 |
Social defeat is induced by physical and psychological stresses, and implicates the cerebral cortex and multiple brain regions of the limbic system [6–8]. Among these regions, the basolateral amygdala (BLA) establishes and consolidates conditioned fears and harmful stimulations. The establishment of conditioned defeat is impaired by infusing the BLA with anisomycin to inhibit protein synthesis. The consolidation of conditioned defeat is enhanced by overexpressing the cyclic adenosine monophosphate (cAMP) response element binding protein in the BLA through a viral vector [9–11]. Therefore, fear consolidation involves a new type of protein synthesis and the formation of neural microcircuit in the BLA. The medial prefrontal cortex (mPFC) is associated with the motivation and the integration in the development of depression and autism. On the one hand, stress stimulation inhibits the mPFC. On the other hand, mPFC activity caused by optogenetics or deep brain stimulation alleviates the symptoms of depression, and which partially contributes to fear extinction through unclear mechanisms [12, 13]. The hippocampus plays pivotal roles in regulating social behavior. Significant evidence indicates that the dorsal and ventral hippocampus (vHIP) play different roles in processing fear and harmful information from context and conditional stimulation . The bidirectional neural circuit between the vHIP and the hypothalamus integrates harmful information and then determines the vulnerability or resilience toward stress stimulation .
|
review
| 99.7 |
The severity of social defeat is determined by the strength and duration of the stimuli. Although different parameters have been considered in establishing rodent models of social defeat, the key criteria are based on common symptoms, including social avoidance, social fear and social withdrawal [16–18]. Social defeat is established in adult rodent through one or more harmful stimulation, followed by gradual recovery through stress removal. The appropriate social strategy is selected according to past experience and learning ability, involving the acquisition, consolidation, retrieval and extinction of social information under specific conditions. Nevertheless, the duration of social defeat is determined by different behavioral paradigms. The changes in the behavior and responses of brain regions are difficult to correlate with one another. Literature has focused on the formation of social defeat and its mechanisms. Few studies have investigated the process of social defeat from establishment to extinction. Our work aims to explore the time characteristics of the consolidation and extinction of acute social defeat and the simultaneous responses of BLA, vHIP, and mPFC. We utilized the resident–intruder paradigm to establish an animal model of acute social defeat. The duration of the consolidation and extinction of social defeat were evaluated. The temporal features of the responses of BLA, vHIP, and mPFC to acute social defeat were also investigated by quantifying the expression of immediate early genes (IEGs), namely, zif268, arc, and c-fos, which are indicators of neural activity.
|
study
| 100.0 |
Muscimol (Mus, cat. G019–5 Mg) and rabbit polyclonal anti-c-fos (cat. HPA018531) were obtained from Sigma (St. Louis, MO, USA). Mouse monoclonal anti-c-fos (cat. Sc8047) and GAPDH (cat. Sc-69778) were obtained from Santa Cruz (Santa Cruz, CA, USA). Rabbit polyclonal anti-parvalbumin antibody (cat. bs-1299R) was purchased from Bioss (Bioss technology company, Beijing, China). Based on the sequences retrieved from a gene bank, Mouse zif268 (accession: NM007913) and arc (accession: NM001276684) primers were designed and produced by Takara Biotechnological Company (Takara Biotech, Co., Ltd., Dalian, China). Kits (cat. 9108) for total RNA extraction were purchased from Takara Biotechnological Company (Takara Biotech, Co., Ltd., Dalian, China). SYBR Green PCR Master Kits (cat. A6001) were purchased from promega (Promega Corporation, USA).
|
other
| 99.7 |
Male C57BL/6 mice weighing approximately 20–22 g and male Kunming (KM) mice weighing approximately 35–40 g were obtained from the Experimental Center of Lanzhou University. Animals were reared in a clean house under a 12-h light/dark cycle, temperature of 22–25 °C, and humidity of 50–60%. Mice were fed with regular diet and purified water ad libtum. All experiments were conducted in accordance with the guidelines of the Policies Governing the Use of Live Vertebrate Animals of the Lanzhou University.
|
other
| 98.75 |
One KM mouse raised alone in a cage for 2 weeks was designated as the resident. The C57BL/6 mice were randomly assigned to a control without the social attack (n = 12) and social defeat with the social attack according to the previous methods . In brief, a C57BL/6 mouse was introduced as the intruder. For 10 min, the C57BL/6 mouse was exposed to the cage with the KM mouse. The aggressive attack was stopped using a perforated plastic barrier, which allows for continued visual, auditory and olfactory contact. After 6 h, the barrier was removed and physical contact was allowed for 10 min. After the second attack, the C57BL/6 mouse was placed back in its home cage. Cumulated frequency of attack is about 40 times per mouse. Mice in social defeat were divided into subgroups on the basis of the designated time at which the animals were sacrificed after social aggression (1, 3, 7, and 15 days; n = 12 per subgroup).
|
study
| 100.0 |
C57BL/6 mice were anesthetized with isoflurane after the completion of social aggression. Mus (40 ng/0.25 µl) or an equal volume of saline was bilaterally injected into the BLA (bregma coordinates: AP − 1.7 mm; ML ± 3.3 mm; DV − 3.8 mm from the brain surface). After 3 days, mice were sacrificed following behavioral examination.
|
study
| 99.94 |
Social fear and avoidance were assessed using the modified open-field system . Briefly, two 9 cm × 9 cm mesh enclosures were placed at the opposite sides of the open field. One enclosure contained a novel KM mouse as the social target, and another enclosure did not contain a mouse as the social un-target. The experimental mouse was allowed to freely explore a 50 cm × 50 cm arena containing the target and un-target enclosures. The time spent on the social interaction zone (14 cm × 26 cm) surrounding the target enclosure and the reference zones (14 cm × 26 cm) surrounding the un-target enclosure was measured using a video tracking system (TM-vision, Chengdu Techman Software Co., Ltd., Chengdu, China).
|
study
| 99.94 |
This test consisted of a 2-bottle procedure in which mice were given the choice between consuming water or a 1% sucrose solution according to previously described method with modification . In brief, animals were singly housed in a cage that had two drinking bottles. One of the bottles had water, while the other bottle had a 1% sucrose solution. Water and sucrose consumption was measured the following day (8:00 A.M.). The position of the sucrose bottle was counterbalanced (left versus right) across the different cages to control for potential side-preference bias.
|
study
| 100.0 |
The marble-burying test was performed according to previously described methods with minor modifications . Briefly, the apparatus consisted of a plastic box (15 × 10 × 10 cm) and 9 clean glass marbles (10 mm diameter) that were evenly spaced (4 cm apart) on sawdust (2 cm deep). The number of marbles that were at least 2/3 buried after 5 min was recorded.
|
study
| 99.9 |
Following behavioral testing, mice were returned to their home cages. After 2 h, mice were deeply anesthetized with isoflurane and transcardially perfused with 20 ml of heparinized saline solution and then with 30 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 and 4 °C. Brains were harvested and stored in the same fixative for 90 min at 4 °C followed by at least 24 h of immersion in 20% sucrose solution in 0.1 M PB with 4% paraformaldehyde. Coronal Sects. (30 µm thickness) were obtained using a cryostat microtome. Sections containing the BLA were mounted onto glass slides and counterstained with Cresyl Violet to precisely locate the microinjection site under light microscopy. The remaining sections were used for immunohistochemistry.
|
study
| 99.94 |
c-Fos immunolabelling was performed using a rabbit polyclonal c-Fos antibody. In brief, sections were quenched in 0.3% hydrogen peroxide (H2O2) for 20 min to remove endogenous peroxidase activity. After serial washing in 0.01 M phosphate- buffered saline (PBS), sections were incubated in phosphate-buffered saline containing rabbit anti c-fos antibody (1:200), 0.3% Triton X, and 0.1% bovine serum albumin (BSA) for 24 h at 4 °C. The incubated sections were then washed and incubated for 90 min in biotinylated goat-anti-rabbit antisera (1:200), followed by washing in 0.01 M PBS. Sections were then incubated in Strep-avidin–biotin–peroxidase complex (ABC) (1:200) for 90 min and then immersed in 0.02% 3,3-diaminobenzidine (DAB) containing 0.01% H2O2 in 0.01 M PBS until a brown reaction product developed.
|
study
| 99.9 |
Photomicrographs were taken with an Olympus microscope BX3 and Olympus DP73 digital camera. Regions of interest were defined in accordance with the mouse brain atlas based on specific landmarks that comprise cellular groups. The anterior–posterior (AP) level from the bregma of the analyzed regions was as follows: BLA (AP: − 0.6 mm to − 0.26 mm), vHIP (AP: -2.8 mm to -3.8 mm) and mPFC (AP: 2.34 mm–1.34 mm). c-Fos immunoreactive profiles were captured from a fixed area under 200 × magnification in at least 3 sections per region per mouse and quantified using ImageJ software (NIH, Bethseda, MD). Given that c-Fos expression levels did not differ between the left and right sides regions in response to any of the experimental procedures, counts were averaged to yield the mean number of c-fos-positive profiles per region per animal.
|
study
| 100.0 |
Following behavioral testing, mice were returned to their home cages. After 2 h, mice were deeply anesthetized with isoflurane and sacrificed. The mouse skull was opened and whole brain was removed. Fresh tissues of BLA, vHIP and mPFC were isolated from the corresponding coronal sections under the stereomicroscope. Total RNA was extracted from the BLA, vHIP or mPFC using RNAiso plus reagent (Takara Biotech, Co., Ltd., Dalian, China) in accordance with the manufacturer’s instructions. DNA contamination was removed with RNase-free DNase. cDNA was synthesized from 1 µg of RNA with M-MuLV reverse transcriptase and random hexamer following the manufacturer’s instructions (Fermentas, Burlington, Canada). Quantitative real-time PCR (Q-RT-PCR) was performed using PIKoREAl96 detector (Thermo Scientific, USA). The mRNA levels of zif268 and arc in triplicate samples of reverse-transcribed cDNA were checked with SYBR Green PCR Master Kit (Promega Corporation, USA) in accordance with the manufacturer’s instructions. The primers for mouse zif268 were 5′-CGAACAACCCTATGAGCACCTG-3′ (forward) and 5′-GGCTGGGATAACTCGTCTCCAC-3′ (reverse). The primers for mouse arc were 5′-GCCAAACCCAATGTGATCCTG-3′ (forward) and 5′-CTGCTTGGACACTTCGGTCAAC-3′ (reverse). The mouse gapdh primers were 5′-GCGAGACCCCACTAACATCAA-3′ (forward) and 5′-GTGGTTCACACCCATCACAAA-3′ (reverse). The assays were initiated for 5 min at 95 °C, 40 cycles of 15 s at 94 °C, and 1 min at 60 °C. The threshold cycles of the target gene and gapdh were calculated. The amplification of zif268 and arc cDNA was normalized to the expression of gapdh. The relative mRNA expression levels of zif268 and arc were calculated using the 2ΔCT method.
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Following behavioral testing, mice were returned to their home cages. After 2 h, mice were deeply anesthetized with isoflurane and sacrificed. The mouse skull was opened and whole brain was removed. Fresh tissues of BLA, vHIP and mPFC were isolated from the corresponding coronal sections under the stereomicroscope. Total proteins were extracted from BLA, vHIP or mPFC samples using RIPA buffer that contained protease inhibitors. The extracted proteins (50 g) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel, and then transferred onto polyvinylidene fluoride membranes. The membranes were blotted with anti-c-Fos (1:1000), anti-GAPDH (1:5000), and horseradish peroxidase-conjugated second antibodies (1:5000). Immunoreactive protein bands were visualized by enhanced chemiluminescence using the Bioanalytical imaging system (Azure Biosystems, INC, USA).
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The data were expressed the mean ± SEM. Statistical analysis was performed using SPSS statistical program, version 17.0. The difference between the two groups was analyzed by Student’s t test, whereas that among three or more groups was analyzed by one-way analysis of variance with least significant difference test. A difference with p < 0.05 was considered statistically significant.
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| 99.44 |
Spontaneous exploration behavior was examined using the open-field test. As shown in Fig. 1c, the freezing time of the intruder mice increased dramatically at days 1 and 3 after stimulation compared with the control mice (F = 4.46, p < 0.01). At day 7 after stimulation, the freezing time of the intruder mice was shorter than that at day 1 after stimulation (F = 4.46, p < 0.05) but was longer than that of the control mice (F = 4.46, p < 0.05). The freezing time of the intruder mice at day 15 after social stimulation was the same as that of the control mice (F = 4.46, p > 0.05). The above data suggests that aversive information from acute stimulation is maintained at least for 7 days before gradually diminishing. The intruder mice recovered their exploratory behavior at 15 days after social defeat. The time spent on the social interaction zone is shown in Fig. 1b, d. At day 1 and day 3 after social aggression, intruder mice spent less time on the interaction zone compared with the control mice (F = 8.98, p < 0.01). On day 7 after stimulation, intruder mice spent more time on the interaction zone than on days 1 and 3 (F = 8.98, p < 0.05) but still spent less on the interaction zone than control mice (F = 8.98, p < 0.05). On day 15 after stimulation, the intruder mice spent the same time on the interaction zone as control mice (F = 8.98, p > 0.05). These data suggest that information for social defeat stress from acute social aggression is maintained for 7 days before diminishing.Fig. 1Behavioral changes after acute social aggressive stimulation. a Diagram of social aggression and social behavioral test. Social aggression is based on the resident (KM mouse)—intruder (C57B6/L mouse) paradigm. b Representative images of the track during social interaction. c Freezing time in the open field. On days 1 and 3 after stimulation, the freezing time of intruder mice was longer than that of control mice. On day 7 after stimulation, the freezing time of intruder mice was shorter than that at day 1 after stimulation but remained longer than that of control mice, one way ANOVA with Tukey’s test was used, F = 4.46, *p < 0.05, **p < 0.01 compared with control, n = 12. d Time spent in the social interaction zones. On days 1 and 3 after stimulation, intruder mice spent less time in the interaction zone than the control mice. On day 7 after stimulation, intruder mice spent more time in the interaction zone than at days 1 and 3 after stimulation but still spent less time in the interaction zone than control mice, one way ANOVA with Tukey’s test was used, F = 8.98, *p < 0.05, **p < 0.01 compared with control, n = 12
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Behavioral changes after acute social aggressive stimulation. a Diagram of social aggression and social behavioral test. Social aggression is based on the resident (KM mouse)—intruder (C57B6/L mouse) paradigm. b Representative images of the track during social interaction. c Freezing time in the open field. On days 1 and 3 after stimulation, the freezing time of intruder mice was longer than that of control mice. On day 7 after stimulation, the freezing time of intruder mice was shorter than that at day 1 after stimulation but remained longer than that of control mice, one way ANOVA with Tukey’s test was used, F = 4.46, *p < 0.05, **p < 0.01 compared with control, n = 12. d Time spent in the social interaction zones. On days 1 and 3 after stimulation, intruder mice spent less time in the interaction zone than the control mice. On day 7 after stimulation, intruder mice spent more time in the interaction zone than at days 1 and 3 after stimulation but still spent less time in the interaction zone than control mice, one way ANOVA with Tukey’s test was used, F = 8.98, *p < 0.05, **p < 0.01 compared with control, n = 12
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Immediate early genes, including zif268, arc and c-fos were used as indices for the evaluation of neural activity. We detected the expression levels of these genes through Q-RT-PCR, Western blotting, and immunohistochemistry. First, we analyzed the mRNA levels of zif268 and arc by using Q-RT-PCR. As shown in Fig. 2a (F = 21.5), b (F = 26.7), the mRNA levels of zif268 and arc in intruder mice gradually increased at day 1, peaked at day 3, decreased at day 7, and were the same as those in control mice at day 15 after stimulation. Second, we examined c-Fos protein levels by using Western blotting. As shown in Fig. 2c, d (F = 5.6), c-Fos expression in intruder mice dramatically increased at day 3 and then slightly decreased at day 7. c-Fos protein levels in intruder mice on days 3 and 7 after stimulation were higher than those in control mice. c-Fos positive cells were labeled and counted as described in “Methods” section. As shown in Fig. 2e, f (F = 16.357), the number of c-Fos positive cells dramatically increased at day 3 after stimulation and then decreased. The above data suggests that aggressive stimulation facilitates BLA activation at the beginning of the stimulus (from day 1 to day 7). This response then diminished during the later stages of the stimulus (from day 7 to day 15).Fig. 2Response of BLA to social defeat stress. a mRNA levels of zif 268 and arc in BLA tissues gradually increased at day 1 after stimulation, peaked at day 3 after stimulation, and decreased at day 7 after stimulation compared with control mice, one way ANOVA with Tukey’s test was used, F = 21.5, *p < 0.05, **p < 0.01 compared with the control, n = 8. b mRNA levels of arc in BLA tissues gradually increased at day 1 after stimulation, peaked at day 3 after stimulation, and decreased at day 7 after stimulation compared with control mice, one way ANOVA with Tukey’s test was used, F = 26.7, *p < 0.05, **p < 0.01 compared with the control, n = 8. c Representative image of the Western blot of c-Fos. d Statistical analysis of c-Fos expression, one way ANOVA with Tukey’s test was used, F = 5.6, *p < 0.05, **p < 0.01 compared with the control, n = 8. e Representative images of c-Fos staining in BLA. Squares in the upper images indicate areas that are shown magnified in the bottom images. Blue arrows indicate c-Fos-positive cells. f Cell count in the BLA, one way ANOVA with Tukey’s test was used, F = 16.357, **p < 0.01 compared with the control, n = 4
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Response of BLA to social defeat stress. a mRNA levels of zif 268 and arc in BLA tissues gradually increased at day 1 after stimulation, peaked at day 3 after stimulation, and decreased at day 7 after stimulation compared with control mice, one way ANOVA with Tukey’s test was used, F = 21.5, *p < 0.05, **p < 0.01 compared with the control, n = 8. b mRNA levels of arc in BLA tissues gradually increased at day 1 after stimulation, peaked at day 3 after stimulation, and decreased at day 7 after stimulation compared with control mice, one way ANOVA with Tukey’s test was used, F = 26.7, *p < 0.05, **p < 0.01 compared with the control, n = 8. c Representative image of the Western blot of c-Fos. d Statistical analysis of c-Fos expression, one way ANOVA with Tukey’s test was used, F = 5.6, *p < 0.05, **p < 0.01 compared with the control, n = 8. e Representative images of c-Fos staining in BLA. Squares in the upper images indicate areas that are shown magnified in the bottom images. Blue arrows indicate c-Fos-positive cells. f Cell count in the BLA, one way ANOVA with Tukey’s test was used, F = 16.357, **p < 0.01 compared with the control, n = 4
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| 100.0 |
zif268, arc mRNA were analyzed by Q-RT-PCR. As shown in Fig. 3a (F = 22.233), b (F = 23.655), the levels of these genes decreased on days 1 and 3 after stimulation and increased at days 7 and 15 after stimulation. We also analyzed the expression of c-Fos protein. Similar to those of zif268 and arc, the expression of c-Fos protein decreased at day 3 and increased at day 15 after stimulation (Fig. 3c, d, F = 4.73). c-Fos-positive cells were labeled and counted as described in "Methods" section. As shown in Fig. 3e, f (F = 5.287), the number of c-Fos-positive cells in intruder mice decreased significantly at days 3 and 7 after stimulation compared with those in the control mice, and then increased to the same levels as those in the control mice at day 15 after stimulation. These results suggest that vHIP is inhibited by aggressive stimulation at the beginning of aggression (from day 1 to day 3), and then gradually recovered (from day 7 to day 15).Fig. 3Response of vHIP to social defeat stress. a mRNA levels of zif268 in vHIP tissues from intruder mice decreased at days 1 and 3 after stimulation and increased at days 7 and 15 after stimulation compared with those in the control mice, one way ANOVA with Tukey’s test was used, F = 22.233, p < 0.05, **p < 0.01 compared with the control, n = 8. b mRNA levels of arc in vHIP tissues from intruder mice decreased at days 1 and 3 after stimulation and increased at days 7 and 15 after stimulation compared with those in the control mice, one way ANOVA with Tukey’s test was used, F = 23.655, p < 0.05, ** p < 0.01 compared with the control, n = 8. c Representative Western blot image of c-Fos. d Statistical analysis of c-Fos expression, one way ANOVA with Tukey’s test was used, F = 4.73, *p < 0.05, compared with the control, n = 8. e The representative images of c-Fos staining in vHIP. Squares in the upper images indicate areas that are shown magnified in the bottom images. Blue arrows indicate c-Fos-positive cells. f Cell counts in the vHIP, one way ANOVA with Tukey’s test was used, F = 5.287, *p < 0.05 compared with control, n = 4
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Response of vHIP to social defeat stress. a mRNA levels of zif268 in vHIP tissues from intruder mice decreased at days 1 and 3 after stimulation and increased at days 7 and 15 after stimulation compared with those in the control mice, one way ANOVA with Tukey’s test was used, F = 22.233, p < 0.05, **p < 0.01 compared with the control, n = 8. b mRNA levels of arc in vHIP tissues from intruder mice decreased at days 1 and 3 after stimulation and increased at days 7 and 15 after stimulation compared with those in the control mice, one way ANOVA with Tukey’s test was used, F = 23.655, p < 0.05, ** p < 0.01 compared with the control, n = 8. c Representative Western blot image of c-Fos. d Statistical analysis of c-Fos expression, one way ANOVA with Tukey’s test was used, F = 4.73, *p < 0.05, compared with the control, n = 8. e The representative images of c-Fos staining in vHIP. Squares in the upper images indicate areas that are shown magnified in the bottom images. Blue arrows indicate c-Fos-positive cells. f Cell counts in the vHIP, one way ANOVA with Tukey’s test was used, F = 5.287, *p < 0.05 compared with control, n = 4
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Levels of zif268 and arc mRNA were analyzed by Q-RT-PCR. As shown in Fig. 4a (F = 99.345), b (F = 43.18), the levels of these genes decreased on days 1, 3, 7 and 15 after stimulation. The expression of c-Fos protein decreased at days 1 and 3 and increased at day 7 after stimulation (Fig. 4c, d, F = 11.4). c-Fos positive cells were labeled and counted as described in "Methods" section. As shown in Fig. 4e, f (F = 23.965), the number of c-Fos-positive cells in intruder mice decreased significantly at days 3 and 7 after stimulation compared with those in the control mice, and then increased to the same levels as those in the control mice at day 15 after stimulation.Fig. 4Response of mPFC to social defeat stress. a mRNA levels of zif268 in mPFC tissues from intruder mice decreased at days 1, 3, 7 and 15 after stimulation compared with the control mice, one way ANOVA with Tukey’s test was used, F = 99.345, **p < 0.01 compared with the control, n = 8. b mRNA levels of arc in mPFC tissues from intruder mice decreased at days 1, 3, 7 and 15 after stimulation compared with the control mice, one way ANOVA with Tukey’s test was used, F = 43.18, **p < 0.01 compared with the control, n = 8. c Representative Western blot image of c-Fos. d Statistical analysis of c-Fos expression, one way ANOVA with Tukey’s test was used, F = 11.4, *p < 0.05, compared with the control, n = 8. e The representative images of c-Fos staining in mPFC. Squares in the upper images indicate areas that are shown magnified in the bottom images. Blue arrows indicate c-fos-positive cells. f Cell counts in the mPFC, one way ANOVA with Tukey’s test was used, F = 23.965, *p < 0.05, **p < 0.01 compared with the control, n = 4
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| 100.0 |
Response of mPFC to social defeat stress. a mRNA levels of zif268 in mPFC tissues from intruder mice decreased at days 1, 3, 7 and 15 after stimulation compared with the control mice, one way ANOVA with Tukey’s test was used, F = 99.345, **p < 0.01 compared with the control, n = 8. b mRNA levels of arc in mPFC tissues from intruder mice decreased at days 1, 3, 7 and 15 after stimulation compared with the control mice, one way ANOVA with Tukey’s test was used, F = 43.18, **p < 0.01 compared with the control, n = 8. c Representative Western blot image of c-Fos. d Statistical analysis of c-Fos expression, one way ANOVA with Tukey’s test was used, F = 11.4, *p < 0.05, compared with the control, n = 8. e The representative images of c-Fos staining in mPFC. Squares in the upper images indicate areas that are shown magnified in the bottom images. Blue arrows indicate c-fos-positive cells. f Cell counts in the mPFC, one way ANOVA with Tukey’s test was used, F = 23.965, *p < 0.05, **p < 0.01 compared with the control, n = 4
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Mus, as GABAA receptor agonist were microinjected to BLA immediately after social aggression. Interestingly, on day 3 after microinjection with Mus or saline, the freezing time of the mice in the Mus-group was lower than that of the mice in the saline-group (Fig. 5c, p < 0.01). Also, the mice in the Mus-group spent more time on the interaction zone than the mice in the saline-group (Fig. 5b, d, p < 0.05). To identify whether social defeat accompanied depression or anxiety, sucrose preference and marble burying test were performed before test of social interaction. The results showed Mus injection increased sucrose preference (Fig. 5e, p < 0.05), but no significant effects on marble burying (Fig. 5f, p > 0.05), which suggested social defeat induced by acute social aggression accompanied depression-like behavior, but not anxiety.Fig. 5Effects of Mus microinjection to the BLA on social defeat stress. a Diagram of experimental flow. b Representative images of the moving track during social interaction. c Freezing time of intruder mice during a 5-min open-field test at day 3 after treatment. d Time spent in the social interaction zone at day 3 after treatment, compared with saline injection. e Sucrose reference at day 3 after treatment. f Marble burying test at day 3 after treatment. Independent student t test was used, *p < 0.05, **p < 0.01 compared with the saline-group, n = 8
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| 100.0 |
Effects of Mus microinjection to the BLA on social defeat stress. a Diagram of experimental flow. b Representative images of the moving track during social interaction. c Freezing time of intruder mice during a 5-min open-field test at day 3 after treatment. d Time spent in the social interaction zone at day 3 after treatment, compared with saline injection. e Sucrose reference at day 3 after treatment. f Marble burying test at day 3 after treatment. Independent student t test was used, *p < 0.05, **p < 0.01 compared with the saline-group, n = 8
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| 100.0 |
Mus were microinjected to BLA immediately after social aggression. At day 3 after social aggression, animals were sacrificed at 2 h after social interaction test. We quantified the expression levels of zif268 and arc in the BLA, vHIP and mPFC. As shown in Fig. 6a, b, the expression levels of zif268 and arc significantly decreased in the BLA and increased in the vHIP of the Mus-group compared with those in the saline-group (p < 0.05). In mPFC, no difference was observed between Mus-treated group and saline-treated group. c-Fos immunochemistry was performed to evaluated neural activity following social interaction test. In vHIP, numbers of c-Fos-positive cells increased in Mus-treated group compared with the saline-treated group (Fig. 6c, d, p < 0.05). In mPFC, numbers of c-Fos-positive cell also increased in Mus-treated group compared with the saline-treated group (Fig. 6e, f, p < 0.05).Fig. 6Effects of Mus microinjection to the BLA on IEGs expression. a zif268 mRNA expression levels in the BLA, vHIP and mPFC. b arc mRNA expression in the BLA, vHIP and mPFC. c Representative images of c-Fos in vHIP. d Numbers of c-Fos-positive cell in vHIP were calculated and statistically analyzed. e Representative images of c-Fos in mPFC. f Numbers of c-fos-positive cell in mPFC were calculated and statistically analyzed. Independent student t test was used, *p < 0.05 compared with the saline-group, n = 4
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| 100.0 |
Effects of Mus microinjection to the BLA on IEGs expression. a zif268 mRNA expression levels in the BLA, vHIP and mPFC. b arc mRNA expression in the BLA, vHIP and mPFC. c Representative images of c-Fos in vHIP. d Numbers of c-Fos-positive cell in vHIP were calculated and statistically analyzed. e Representative images of c-Fos in mPFC. f Numbers of c-fos-positive cell in mPFC were calculated and statistically analyzed. Independent student t test was used, *p < 0.05 compared with the saline-group, n = 4
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| 100.0 |
We found that social defeat induced by acute aggression is characterized by social fear and avoidance, and is accompanied by a depression-like behavior. Behavioral test revealed that social fear was severe on days 1 and 3 and gradually diminished on days 7 and 15 after aggression. On days 1 and 3 after aggression, the expression of IEGs (including zif268, arc, and c-Fos) increased in the BLA but decreased in the vHIP and mPFC. On days 7 and 15 after aggression, the expression of IEGs decreased in the BLA but increased in the vHIP. Thus, BLA played an important role in establishing social defeat. Equal volumes of Mus and saline were microinjected into the bilateral BLA immediately after aggression to verify this hypothesis. On day 3 after aggression, social fear and freezing behavior were more significantly ameliorated by Mus treatment than by saline treatment. In addition, Mus treatment reduced the expression of IEGs in the BLA but increased that in the vHIP. The amygdala is critical in mediating fear- and anxiety-related behavior and is the key site for the acquisition and storage of fear memory. The plasticity of the sensory inputs from the thalamus and the cortical areas to the projection neurons in the BLA is the core mechanism underlying Pavlovian fear conditioning [23–25]. The associations between conditioned and unconditioned stimuli are formed through this mechanism. According to the neural circuit model of the amygdala function, fear output is generated by the associative information conveyed from the BLA by internuclear excitatory projections from the basal and basomedial nuclei to the medial region of the central amygdala. This passive information transfer model is currently being challenged because growing evidence indicates that the BLA and central amygdala can independently mediate parallel or additional associative functions under aversive conditioning [26, 27]. Another emerging evidence is that fear acquisition is controlled by the plasticity of the inhibitory synapses and the interneurons in the amygdala . On the one hand, regulating the inhibition in the BLA and the specific inhibitory synapses on principal neurons in the BLA is associated with the behavioral suppression of fear following the extinction of learning. On the other hand, the activity of local GABAergic neurons is regulated by the plasticity of their excitatory inputs in the BLA and the central lateral amygdala . A recent study revealed that specific local interneurons in the BLA were differentially recruited during conditioned and un-conditioned stimulation associations, thereby mediating the inhibition and disinhibition of distinct subcellular domains for controlling fear learning. Therefore, multiple plastic systems, particularly those integrating GABAergic neurons, can collectively encode aspects of the stimulus associations in the amygdala. The local interneurons in the BLA are heterogeneous interneurons displaying different morphological, electrophysiological, and neurochemical characteristics. Parvalbumin positive interneurons constitute approximately 40% of these interneurons and exert robust peri-somatic inhibitory effects on the projection neurons [30, 31]. We found that the ratio of c-Fos to parvalbumin double labeled cells in the BLA was approximately 20% of the total c-Fos immunopositive cells on day 3 after social aggression (Additional file 1: Figure S1). These findings suggested that the interneurons in BLA are involved in the regulation of social defeat.
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| 99.94 |
The extinction of fear memory is one treatment method for post-traumatic stress disorder, because the establishment of a novel social interaction is accompanied by the extinction of previous social fear. In this study, the recovery due to social interaction was observed on day 15 after aggression, suggesting that the previous aversive stimulation was gradually diminished. Researchers have recently used a condition stimulus memory retrieval–extinction procedure to prevent fear recovery after extinction in rats and humans [32–35]. Consolidation refers to the process wherein memories are retained after their retrieval and destabilization. Destabilization refers to the return of a memory to a labile phase after memory retrieval. Researchers have inferred that memory consolidation is hampered when the expression of the conditioned responses is disrupted by post-retrieval neural pharmacological manipulations within a specific time interval (i.e., a “reconsolidation window” of up to 2 h post retrieval. This strategy effectively promoted the extinction of aversive memory, which aided in ameliorating the symptoms of mood disorders [36–38]. Our results suggested that aversive memory became extinct after 15 days without additional social defeat. Once the aversive information is forgotten, the learning ability for a novel stimulation is recovered. Another recent study found that autism is associated with genetic variation and the copy number deletion of P-Rex1, which encodes the phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor1. The genetic deletion or knockdown of P-Rex1 in the CA1 region of the mouse hippocampus produced autism-like behaviors, such as impaired social interactions, reversal learning deficits in the water maze, and extinction-resistant memory of fear conditioning [39, 40]. These behavioral changes were possibly due to impaired active forgetting or, in a general sense, the failure to update memories. Context recognition involves hippocampal function. The inhibition of the vHIP induced by social aggression disturbs the recognition of novel context and social objects at an early stage (on days 1 and 3 after social defeat). The hippocampal response to novel stimulation resulted in the recovery of the learning ability at a later stage (on days 7 and 15 after social defeat).
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study
| 99.94 |
The mPFC is associated with motivation as the center of emotion integration. A previous study posited that the activation of mPFC diminished social defeat . Deep brain stimulation in the mPFC ameliorated the symptoms of patients suffering from severe depression . The social avoidance of the intruder mice reduced the expression of IEGs in the mPFC for novel contexts and social objects at an early stage of social defeat. The social interaction of the control mice led to the high expression of IEGs in the mPFC for novel social objects. The inhibition of the BLA by Mus treatment increased the social interaction and exploration time for novel contexts, suggesting that BLA establishes and consolidates social information. Stress recovery was aided by inhibiting the function of BLA after an aversive stimulation at an early stage. Disturbances at critical times affect the efficacy of the consolidation or extinction of the aversive stimulation. Consolidation and extinction are based on synaptic plasticity and new protein synthesis, both of which are dependent on the duration of the neural activity. Therefore, the time window of a treatment is important. Salinas reported that training-related IGF-II-dependent memory enhancement is restricted to a temporal window of less than a day. However, the enhancing effect re-emerges at a later time if IGF-II is combined with memory retrieval, which reactivates the memory and induces reconsolidation. The IGF-II effect following retrieval is also temporally limited and restricted to a temporal window that overlaps with the reconsolidation-sensitive period of inhibition avoidance . For the retrieval-induced memory fragility and the IGF-II-dependent enhancement, both of them require new protein synthesis,but in different brain regions, such as the amygdala in the former and the hippocampus in the latter. Therefore, social fear memory was in a sensitive period during the first 3 days after aggression. When activated, this memory could either be significantly weakened or enhanced. These findings suggested that reconsolidation is a lingering consolidation process [44, 45].
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| 99.94 |
Although the methodology was limited, our data revealed the duration of social defeat from consolidation to extinction after an acute social aggression. In the future, we will explore the relationship between the responses of the BLA and the vHIP toward social defeat, particularly the functions of the neural circuit between the BLA and the vHIP in recognizing social contexts and objects. We aim to use extinction training and noninvasive treatment to alleviate social defeat and explore the mechanisms underlying the temporal windows of consolidation and extinction.
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| 100.0 |
Acute social defeat was induced by using a social aggressive attack according to the intruder–resident paradigm. The social behavior of the intruder mice toward novel contexts and objects was measured on days 1, 3, 7, and 15 after aggression. The expression of IEGs in the BLA, vHIP, and mPFC was analyzed at 2 h after behavioral test. The results indicated that social fear and avoidance were significant on days 1–3 after aggression and were accompanied by a high expression of IEGs in the BLA and a low expression of IEGs in the vHIP and mPFC. On days 7–15 after aggression, the social interaction gradually increased and was accompanied by a low expression of IEGs in the BLA and a high expression of IEGs in the vHIP. Inhibiting the function of BLA after aggression ameliorated social defeat, which suggested that BLA consolidates social fear. This study posited that social defeat can be treated by suppressing the consolidation or prompting the extinction of aversive stimulations.
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| 100.0 |
Additional file 1: Figure S1. To double-label BLA with c-fos and parvalbumin, BLA sections were stained with parvalbumin and developed with DAB. c-Fos was labeled and developed with DAB-nickel ammonium sulfate. Representative images of c-Fos and parvalbumin double labeling, blue arrows indicate the double labelling cells; black arrows indicate the single labelling cells.
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| 99.94 |
Additional file 1: Figure S1. To double-label BLA with c-fos and parvalbumin, BLA sections were stained with parvalbumin and developed with DAB. c-Fos was labeled and developed with DAB-nickel ammonium sulfate. Representative images of c-Fos and parvalbumin double labeling, blue arrows indicate the double labelling cells; black arrows indicate the single labelling cells.
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| 99.94 |
The spinal cord injury (SCI) is very common and has poor prognosis (Webb et al., 2010). SCI could cause severe damage to motor, sensory, and autonomic nervous system and their functions which may lead to severe disabilities (Coutts and Keirstead, 2008). The pathophysiology of SCI after the primary trauma has a vital role in the initial structural disruption. In addition, the secondary process includes the cascades of cellular and biological disruptions which can lead to long-term spinal deficits (Dasari et al., 2014). Increased oxidative stress (Fatima et al., 2015), redox transcription factors activity as well as elevated expression of inflammatory mediators play important roles (Popovich and Jones, 2003) in the promotion of the secondary process after the initial injury. SCI results in structural deformation such as degeneration of axons, disruption of neural tissue, neural and glial cell death, and demyelination around the lesion site. Axonal regeneration is inhibited by myelin-associated inhibitors in the central nervous system (CNS; Geoffroy and Zheng, 2014) and formation of astrogliosis (Deng et al., 2011). The extent of intrinsic cell renewal alone (Li and Lepski, 2013), even after application of mitogenic agents such as variety of growth factors, is not sufficient enough for substantial recovery following SCI (Chhabra and Sarda, 2015). So, therapeutic approaches such as exogenous cell transplantation should be considered.
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review
| 99.9 |
Human umbilical cord blood stem cells (hUCBSCs) hold great promise for therapeutic repair after SCI (Dasari et al., 2007). The human umbilical cord blood by differentiating into various neural cells can enhance motor and sensory function improvement in the animal models of SCI (Kuh et al., 2005).
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review
| 99.9 |
Transplantation of human umbilical cord blood mononuclear cells into the injured site of spinal cord did not affect the lesion extension. The survival time of transplanted cells in the injured area was 6 weeks after treatment. The transplanted group indicated better functional recovery than the untreated ones (Rodrigues et al., 2012). There is sufficient evidence which prove that stem cell therapy could be effective in spinal cord injuries but a strategy to potentiate this stem cell transplantation results is required (Niapour et al., 2012; Chhabra and Sarda, 2015).
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review
| 99.9 |
As there have been strong interests in finding traditional agents that may help in the prevention of inflammation and disruption of neural tissues in CNS, a well-known herbal drug which has exhibited antioxidant and neuroprotective effects is Melissa officinalis (MO). MO is commonly known as lemon balm (family: Lamiaceae); it is one of the oldest and still the most common medicinal plants (Kamdem et al., 2013). It has been reported that the most commonly known therapeutic properties of MO extract were sedative, antispasmodic, carminative, antibacterial, antiviral, antiinflammatory, antioxidant, as well as neuroprotective (Kamdem et al., 2013). It was previously shown that the effective dose of MO was 150 mg/kg in SCI contusive model, and it was also shown that MO extract was effective in improving motor, sensory, and cellular function after injury (Hosseini et al., 2015). Therefore, the aim of this study is to assess the effectiveness of the MO extract in combination with hUCBSC transplantation after contusive SCI in Wistar rats. It was hypothesized that the combination of MO and hUCBSCs may play a role in preventing harmful effects and neural damage triggered by SCI.
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study
| 99.9 |
After obtaining the approval of the Institutional Review Board of our university, all experiments were carried out in accordance with the Guidelines for the Animal Care and use ethics committee of the Baqiyatallah University of medical sciences. Thirty-six adult male Wistar rats weighing from 190 to 220 g were maintained under standard laboratory conditions. Animals were housed in an environment of 21 ± 2℃ with a relative humidity of 50 to 10% and a 12-hr light–dark cycle. Food and water were always available.
|
study
| 99.9 |
To make SCI, the animals were anesthetized with 80 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride (Alfasan Company, Netherlands) intraperitoneally. Weight-drop contusion method was conducted to induce SCI in rats. The skin and subcutaneous tissues in the thoracolumbar T12-L1 region were incised. After penetration of paravertebral muscle fascia, muscles were peeled laterally using blunt dissection forceps. The spinal cord segment at T12-L1 level was exposed by total laminectomy. The animals were subjected to an impact of 10 g weight (stainless steel rod, 3 mm diameter tip) dropped vertically in the center of the exposed spinal cord from the height of 25 mm (severe; Agrawal et al., 2010). In the sham group, all mentioned procedures were carried out, except the spinal cord contusion. The final procedure was incision suturing (Byrnes et al., 2010).
|
study
| 100.0 |
Core body temperature of animals was maintained at 36.5 to 37.5℃ during and after the study procedures. The rats were treated with gentamicin (40 mg/kg, intramuscular injection; Caspian Tamin Company, Iran) twice a day for the first 3 days as prophylaxis against urinary tract infection. The urinary bladders were pressed three times a day by the time that bladder function returned to normal. The rats were also injected subcutaneously with 25 ml/kg lactated Ringer’s solution (Caspian Tamin Company, Iran) for 2 days after SCI as once a day (Edalat et al., 2013).
|
study
| 100.0 |
The plant was taken from the commercial source. The dried leaves powder of MO was macerated at room temperature in 70% ethanol (1 g/10 ml) and extracted for a week. On the seventh day, the ethanolic extract was refined and the extract was evaporated under reduced pressure to remove the ethanol. The dry extract was suspended in the normal saline and thus alcoholic extract of MO was prepared. We have previously shown that most effective dose of MO in the animal model of SCI is 150 mg/kg. So, in this study, we administered 150 mg/kg of MO (Pereira et al., 2009; Hosseini et al., 2015).
|
study
| 100.0 |
Rats were randomly divided into six groups as follows: Group І: intact group (n = 5), Group ІІ: sham rats were subjected to laminectomy without SCI (n = 5), Group ІІІ: rats were subjected to laminectomy and SCI (n = 5), Group ІV: rats were subjected to laminectomy, SCI, and treated with 150 mg/kg MO (SCI-MO; n = 7), Group V: rats were subjected to laminectomy, SCI, and treated with hUCBSCs (SCI-hUCBSC; (n = 7), Group VІ: rats were subjected to laminectomy, SCI, and treated with combination of 150 mg/kg MO and hUCBSCs (SCI-MO-hUCBSC; n = 7). hUCBSCs were transplanted intraspinally 24 hr after injury. MO was daily injected intraperitoneally into treatment rat groups starting 1 day after injury for 14 days.
|
study
| 100.0 |
Human umbilical cord blood was collected from healthy women aged between 20 and 40 years with informed consent and according to the protocol approved by Institutional Review Board of Baqiyatallah University of Medical Sciences. Thereafter, human umbilical cord blood was transferred into Falcon tube containing phosphate buffered saline (PBS) without Ca2+ or Mg2+ supplemented with 2.5 µg/ml fungizone, 100 µg/ml streptomycin, 100 U/ml penicillin, and 0.5 Mm ethylenediaminetetraacetic acid (All from Merck, Germany). Mononuclear cells were separated utilizing Ficoll-Hypaque (Sigma, St. Louis, MO) density gradient centrifugation and washed out in PBS. Thereafter, the cell pellet in the tube was suspended in Dulbecco's modified Eagle medium and Ham’s F-12 (DMEM-F12) medium supplemented with 10% fetal bovine serum (FBS; All from Sigma) and cultured in tissue culture plates. The cells were kept in the 37℃ incubator with 5% CO2 and saturated humidity. After removing nonadherent cells in the second day of incubation, the culture of adherent cells continued until 70% confluency (Dasari et al., 2007; Faramarzi et al., 2016). At Passage 4, the cells were checked for the properties of mesenchymal stem cells using fibronectin (+), CD44 (+), and CD45 (−) (Santa Cruz Biotechnology, Santa Cruz, CA) immunostaining. The dissociated mesenchymal cells were further dispersed in 10% FBS-DMEM and counted under a microscope with the aid of a hemocytometer. The mesenchymal cells were then utilized directly for cultures or stored in liquid nitrogen for later use.
|
study
| 100.0 |
To enable the visualization of hUCBSCs after their transplantation into the spinal cord, their nuclei were labeled with 5-bromo-2′-deoxyuridine (BrdU). BrdU (Merck, Germany) at the final concentration of 10 mg/ml was added to the culture of the hUCBSCs 24 hr before transplantation. The excess tracer was washed out with PBS and cells were suspended in fresh culture medium to obtain approximately 300,000 cells in 10 μl.
|
study
| 99.94 |
Animals were reanesthetized as described earlier, and the laminectomy site was re-exposed. Sham group animals were injected 24 hr after laminectomy with 9 µl of normal saline by utilizing a10 µl Hamilton syringe (Sigma). The hUCBSCs-treated group was injected 24 hr after injury. The mononuclear cell layer of hUCBSC (3 × 105 ells/µL) in 9 µl of normal saline at a rate of 0.25 µl/min was transplanted into the three sites of lesion area (epicenter, distal, and proximal) at a depth of 1.2 mm. The hUCBSCs were previously labeled with BrdU so as to facilitate the identification of the cells within the subsequent histological specimens. Cyclosporine A (10 mg/kg; (Sigma) was administered as an immunosuppressant for 7 days after transplantation of hUCBSCs (Dasari et al., 2007).
|
study
| 100.0 |
For assessment of neurological function, the Basso–Beattie–Bresnahan (BBB) scale was used for open field motor testing in all rat groups. The BBB scale is a 21-point scale ranging from 0 to 21 (Barros Filho and Molina, 2008), rating locomotion on aspects of hind limb function such as weight support, stepping ability, coordination, and toe clearance (Byrnes et al., 2010). All functional scores were obtained on Days 1, 7, 14, 21, 28, 35, 42, 49, and 56 by two individuals who were blinded to treatment. The final score of each animal was the mean value of both examiners (Hosseini et al., 2015).
|
study
| 100.0 |
Behavioral test for evaluation of sense of pain was performed by means of hot water test for the hind limbs after SCI (scores were obtained on Days 1, 7, 14, 21, 28, 35, 42, 49, and 56). The response to heat stimulation was measured by the latency of hind limb paw withdrawal of hot water at 50℃. Both paws of rats were kept in a hot water container, respectively. For each rat, six trials were obtained (three trials for each paw), and mean of this trials were recorded, and nonresponders were removed from the hot water container after 60 s (Kim et al., 2013; Hosseini et al., 2015).
|
study
| 100.0 |
Spontaneous rest activity was recorded from hind limb flexor muscle bilaterally. Electromyography (EMG) recording was done by 23 gauge needles for 10 s one day prior to sacrifice of animals. The EMG signal was amplified (Grass, Astro-Med, West Warwick, RI), digitized (5 kHz, Digi-data 1322A; Axon instruments, Foster City, CA), and filtered (30–300 Hz; Fouad et al., 2013). After recording, the recruitment index of motor units was acquired via compression of 10 s of recording to 1 s by EMG software. The recruitment index was scored on an ordinal scale (0 to ++++) (Stålberg et al., 1998; Hosseini et al., 2015).
|
study
| 100.0 |
On Day 57, all rats were anesthetized (100 mg/kg sodium pentobarbital, I.P.). Thereafter intracardially perfused with 0.9% saline followed by 10% buffered formalin. A spinal cord segment at the level of T12-L1 was dissected, postfixed in 10% buffered formalin overnight, cryoprotected in 30% sucrose for 48 hr and serially transverse sectioned using a cryostat (B1155800 Sakura) at 10 µm thickness. All sections were processed for hematoxylin and eosin staining and assessed under light microscopy (Byrnes et al., 2010). Standard immunohistochemistry for the glial scar (glial fibrillary acidic protein) and myelination (myelin basic protein [MBP]) was performed for all of the sections. For immunohistochemistry, sections from formalin-fixed, paraffin-embedded spinal cord tissues were dewaxed, rehydrated, and retrieval of antigens was performed. After incubation with 3% H2O2 in methanol, and then normal nonimmune goat serum, the sections were incubated with rabbit antiactive glial fibrillary acidic protein (GFAP) polyclonal antibody and mouse monoclonal MBP primary antibody (Santa Cruz Biotechnology), at a dilution of 1:200 at 4℃ for overnight, followed by biotinylated goat anti-rabbit IgG for 20 min at room temperature, and subsequently incubated with streptavidin–peroxidase (All from Santa Cruz Biotechnology). PBS replaced primary antibody as the negative control. 3,3′-Diaminobenzidine chromogen was applied for visualization of peroxidase activity. Finally, the sections were counterstained with hematoxylin (Fleming et al., 2006; Hosseini et al., 2015).
|
study
| 99.94 |
The lesion area including the cavity and surrounding damaged tissue in area of 3562,500 µm2, was then measured by using an image analyzing software (Motic 2.1, Italy, Cagli); in addition, the number of lower motor neurons in area of 5,700 µm2, the number of positive GFAP astrocyte perikaryon in ventral horn, and area of 35,625 µm2 were measured. Only those cells that showed clearly discernible nucleus were counted. Densities of myelin in dorsal white matter and astrogliosis in the ventral horn of spinal cord were evaluated by using of histolab software (Iran, 1392). Five sections from each case were evaluated, and mean values were obtained for each animal. Cell counting and densitometry analyzes were carried out by two observers who were blind to the specific experimental conditions of the analyzed tissues on images acquired at 40×, 400×, and 1000× magnifications(Bharne et al., 2013).
|
study
| 100.0 |
For electron microscopy, spinal cords from five rats from each treatment group were processed into small 3 mm3 blocks that surrounded the injury epicenter and fixed for 1 hr in a mixture of glutaraldehyde (1.5%) and paraformaldehyde (3%), followed by washing three times in 0.1 M sodium cacodylate and 3 mM CaCl2. Samples were then postfixed in potassium ferrocyanide (0.8%) and osmium tetroxide (1%) for 1 hr followed by 3× washes in 0.1 M sodium cacodylate and 3 mM CaCl2 (All from Sigma). Ensuing a brief dH2O rinse, samples were embedded in Eponate 12 (Pelco, Clovis, CA) and cured at 60℃ for 2 days. Spinal cord sections (80 nm in thickness) corresponding to the site of the lesion were cut on a Reichert Ultracut E with a Diatome diamond knife, collected on formvar-coated 1 × 2 mm2 copper grids, and stained with uranyl acetate followed by lead citrate. Sections were examined on a Hitachi 7600 transmission electron microscope operating at 80 kV. The myelin index (MI) was measured by means of the ratio of axon diameter to axon diameter plus its myelin sheath (Wrathall et al., 1998; Pang et al., 2012).
|
study
| 100.0 |
T12-L1 segments of spinal cord from various groups were homogenized and total RNAs were isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Approximately 1 µg of total RNA from each sample was reverse transcribed into cDNA according to the manufacturer’s instructions using the iScrip™ cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was applied as an internal control. We used the following sequences for the forward and reverse primers:
|
study
| 99.94 |
The housekeeping gene GAPDH was used for normalization of MBP mRNA expression. Samples were subjected to 25 to 35 cycles at 95℃ for 30 s, 60℃ for 30 s, and 72℃ for 1 min on GeneAmp PCR System 9700 (Perkin Elmer, Boston, MA) in 25 µl reaction volumes. After amplification, reverse transcription-polymerase chain reaction products were separated on a 1% agarose gel containing 0.5 mg/ml ethidium bromide. The amplified cDNA fragments were visualized under ultraviolet light (Dasari et al., 2007).
|
study
| 99.94 |
Data obtained from motor and sensory functions at each time point as well as electromyographic activity between different groups were analyzed utilizing two-way analysis of variance (ANOVA). The histomorphometric, immunostaining data, densitometry, and electron microscopic data were analyzed using one-way ANOVA. In both tests, ANOVA was followed by post hoc Bonferroni’s multiple comparison tests. Data were presented as the mean ± SEM and a significance level of .05 was predetermined for all statistical analysis.
|
study
| 100.0 |
Immunocytochemistry examination detected the localization of fibronectin and CD44 on hUCBSCs. The percentage of positivity was 89.67 and 94.78%, respectively. hUCBSCs were negative for the marker CD45 (Figure 1). Furthermore, immunofluorescence studies revealed the presence of transplanted cells in lesion area of the spinal cord (Figure 2). Figure 1.Characterization of hUCBSCs. (a) Cells stain positive for fibronectin (brown). (b) Cells stain positive (brown) for CD44. (c) Cells do not stain for CD45. (d) Cells labeled with BrdU. Note. hUCBSCs = human umbilical cord blood stem cells; BrdU = Br5-bromo-2′-deoxyuridine. Figure 2.Transplanted hUCBS cells in the injured spinal cord, with anti-BrdU antibody as a primary antibody followed by the secondary antibody conjugated with FITC. The figure represents a qualitative feature of the immunostained cells (a). (b) Shows phase contrast of (a) picture. Note. hUCBS = human umbilical cord blood stem; BrdU = Br5-bromo-2′-deoxyuridine; FITC = fluorescein isothiocyanate.
|
study
| 100.0 |
Characterization of hUCBSCs. (a) Cells stain positive for fibronectin (brown). (b) Cells stain positive (brown) for CD44. (c) Cells do not stain for CD45. (d) Cells labeled with BrdU. Note. hUCBSCs = human umbilical cord blood stem cells; BrdU = Br5-bromo-2′-deoxyuridine.
|
other
| 99.9 |
Transplanted hUCBS cells in the injured spinal cord, with anti-BrdU antibody as a primary antibody followed by the secondary antibody conjugated with FITC. The figure represents a qualitative feature of the immunostained cells (a). (b) Shows phase contrast of (a) picture. Note. hUCBS = human umbilical cord blood stem; BrdU = Br5-bromo-2′-deoxyuridine; FITC = fluorescein isothiocyanate.
|
other
| 99.56 |
SCI resulted in immediate paraplegia (loss of hind limb movement); hence the SCI group demonstrated significant changes in locomotion scores in comparison with intact group. hUCBSCs significantly enhanced locomotors function in rats when compared with SCI group. Furthermore, when intraperitoneal MO (150 mg/kg) was added a day after the injury, it significantly improved locomotors function in rats when compared with SCI group. The application of two-way ANOVA showed significant interaction between variables such as hUCBSCs therapy, MO treatment, and time, F(40, 270) = 29.37, p < .001. Application of post hoc Bonferroni’s multiple comparison tests showed significant improvement in motor function following 150 mg/kg MO treatment on Day 35, 42 (p < .01), 49, and 56 (p < .001) and hUCBSCs therapy on Day 14, 21 (p < .05), 28 (p < .01), 35, 42, 49, and 56 (p < .001) in comparison with SCI group. Furthermore, the combination of MO and hUCBSCs significantly enhanced motor function on Day 14, 28 (p < .01), 21, 35, 42, 49, and 56 (p < .001) in comparison with SCI group. There were no significant differences between SCI-MO, SCI-hUCBSC, and SCI-MO-hUCBSC groups (Figure 3). Figure 3.Effect of hUCBSC-MO treatment on motor function after SCI. Intraperitoneal injection of MO (150 mg/kg) was started 1 day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of BBB score ± SEM, (n = 5–7) and analyzed by two-way ANOVA followed by post hoc Bonferroni’s multiple comparison test. ***p < .001 shows significant different between SCI versus intact. ΩΩ, and ΩΩΩ show significant difference between SCI-MO and SCI. Φ, ΦΦ, and ΦΦΦ show significant difference between SCI-hUCBSC and SCI. ##, and ### show significant difference between SCI-MO-hUCBSC and SCI (p < .05, p < .01, and p < .001, respectively). Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; SCI = spinal cord injury; BBB = Basso–Beattie–Bresnahan; ANOVA = analysis of variance.
|
study
| 99.94 |
Effect of hUCBSC-MO treatment on motor function after SCI. Intraperitoneal injection of MO (150 mg/kg) was started 1 day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of BBB score ± SEM, (n = 5–7) and analyzed by two-way ANOVA followed by post hoc Bonferroni’s multiple comparison test. ***p < .001 shows significant different between SCI versus intact. ΩΩ, and ΩΩΩ show significant difference between SCI-MO and SCI. Φ, ΦΦ, and ΦΦΦ show significant difference between SCI-hUCBSC and SCI. ##, and ### show significant difference between SCI-MO-hUCBSC and SCI (p < .05, p < .01, and p < .001, respectively). Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; SCI = spinal cord injury; BBB = Basso–Beattie–Bresnahan; ANOVA = analysis of variance.
|
study
| 100.0 |
Statistical evaluations showed that the mean latency time of response to the painful stimulus decreased significantly in SCI-hUCBSC group when compared with SCI group. When intraperitoneal MO treatment (150 mg/kg) was added a day after the injury, it significantly enhanced sensory recovery in rats when compared with SCI group. The application of two-way ANOVA showed significant interaction between variables including hUCBSCs, MO treatment (150 mg/kg), and time, F(40, 270) = 12.39, p < .001. Application of post hoc Bonferroni’s multiple comparison tests showed significant improvement in sensory function following 150 mg/kg MO treatment on Day 28 (p < .01), 35, 42, 49, and 56 (p < .001) and hUCBSCs therapy on Day 28 (p < .01), 35, 42, 49, and 56 (p < .001) in comparison with SCI group. Moreover, combination of MO and hUCBSCs significantly improved motor function on Day 14 (p < .05), 21 (p < .001), 28, 35, 42, 49, and 56 (p < .001) in comparison with SCI group. There were no significant differences between SCI-MO, SCI-hUCBSC, and SCI-MO-hUCBSC groups (Figure 4). Figure 4.Effect of hUCBSC-MO treatment on sensory function after SCI. Intraperitoneal injection of MO (150 mg/kg) was started 1 day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of latency time ± SEM, (n = 5–7) and analyzed by two-way ANOVA followed by post hoc Bonferroni’s multiple comparison test. ***p < .001 significant difference between SCI versus intact. ΩΩ, and ΩΩΩ show significant difference between SCI-MO and SCI. ΦΦ, and ΦΦΦ show significant difference between SCI-hUCBSC and SCI. #, ##, and ### show significant difference between SCI-MO-hUCBSC and SCI (p < .05, p < .01, and p < .001, respectively). Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; ANOVA = analysis of variance; SCI = spinal cord injury.
|
study
| 99.94 |
Effect of hUCBSC-MO treatment on sensory function after SCI. Intraperitoneal injection of MO (150 mg/kg) was started 1 day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of latency time ± SEM, (n = 5–7) and analyzed by two-way ANOVA followed by post hoc Bonferroni’s multiple comparison test. ***p < .001 significant difference between SCI versus intact. ΩΩ, and ΩΩΩ show significant difference between SCI-MO and SCI. ΦΦ, and ΦΦΦ show significant difference between SCI-hUCBSC and SCI. #, ##, and ### show significant difference between SCI-MO-hUCBSC and SCI (p < .05, p < .01, and p < .001, respectively). Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; ANOVA = analysis of variance; SCI = spinal cord injury.
|
study
| 100.0 |
Although after the application of two-way ANOVA, there was no significant difference between the right and left hind limb. The statistical analysis indicated that the means of recruitment index were significantly increased for left and right hind limbs in SCI-MO, SCI-hUCBSC, and SCI-MO-hUCBSC groups when compared with SCI group, F(5, 60) = 0.01, p < .001. Application of post-hoc Bonferroni’s multiple comparisons test as well as Bartlett’s test for equal variances showed significant improvement in electrophysiological activity of left and right hind limbs following 150 mg/kg of MO extract administration (p < .01), hUCBSC therapy, and hUCBSC-MO treatment (p < 0.001) in comparison with SCI group. There were no significant differences between SCI-MO, SCI-hUCBSC, and SCI-MO-hUCBSC groups (Figure 5). Figure 5.Effect of hUCBSC-MO on electromyographic activity after SCI. Intraperitoneal injection of MO (150 mg/kg) was started a day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of recruitment index ± SEM, (n = 5–7) and analyzed by two-way ANOVA followed by post-hoc Bonferroni’s multiple comparison test. ***p < .001 shows significant difference between SCI versus intact. ##p < .01 and ###p < .001 versus spinal cord injury. Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; SCI = spinal cord injury; SEM = standard error of the mean; ANOVA = analysis of variance.
|
study
| 100.0 |
Effect of hUCBSC-MO on electromyographic activity after SCI. Intraperitoneal injection of MO (150 mg/kg) was started a day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of recruitment index ± SEM, (n = 5–7) and analyzed by two-way ANOVA followed by post-hoc Bonferroni’s multiple comparison test. ***p < .001 shows significant difference between SCI versus intact. ##p < .01 and ###p < .001 versus spinal cord injury. Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; SCI = spinal cord injury; SEM = standard error of the mean; ANOVA = analysis of variance.
|
study
| 100.0 |
In the intact group, spinal cord segments were not damaged in both white and gray matter. Application of one-way ANOVA demonstrated that the mean cavity size in terms of mm2 was significantly reduced in treatment groups, F(5, 30) = 27.95, p < .001. Moreover, post hoc Bonferroni’s multiple comparison test illustrated the significant reduction in the mean cavity area in SCI-MO, SCI-hUCBSC (p < .01), and SCI-MO-hUCBSC (p < .001) groups when compared with SCI group. Furthermore, application of one-way ANOVA showed that mean cavity area in SCI-MO-hUCBSC group was significantly reduced in comparison with SCI-MO and SCI-hUCBSC groups (p < .05; Figure 6). Figure 6.Effect of hUCBSC-MO treatment on cavity formation after SCI. Intraperitoneal injection of MO (150 mg/kg) was started one day after injury and continued once a day for 14 days after injury. Intraspinal grafting of hUCBSCs was started 24 hr after injury. Data are represented as mean of the cavity area ± SEM (n = 5–7) and analyzed by one-way ANOVA followed by post hoc Bonferroni’s multiple comparison test. ***p < .001 shows significant difference between SCI versus intact. ##p < .01, and ###p < .001 versus spinal cord injury. Φ shows significant difference between SCI-MO-hUCBSC and SCI-hUCBSC (p < .05). $ shows significant difference between SCI-MO-hUCBSC and SCI-MO (p < .05, p < .01, and p < .001, respectively). Note. hUCBSCs = human umbilical cord blood stem cells; MO = Melissa officinalis; SCI = spinal cord injury; SEM = standard error of the mean; ANOVA = analysis of variance.
|
study
| 100.0 |
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