This study assesses malaria prevention and treatment behaviour among people living with HIV/AIDS (PLWHA) in Owerri, South Eastern Nigeria. Although Nigeria bears one of the world’s largest burdens of both malaria and HIV, there is almost no research studying how co-infected patients manage their care. We systematically sampled 398 PLWHA receiving care at Imo State Specialist Hospital and the Federal Medical Centre in Owerri to complete a structured, pre-tested questionnaire on malaria care-seeking behaviour. Descriptive statistics were reported and chi-square tests and multivariate logistic regressions were also used. The majority of HIV-infected patients (78.9%) reported having had an episode of suspected malaria quarterly or more often. There was a large variation in care-seeking patterns: on suspicion of malaria, 29.1% of participants engaged in self-medication; 39.2% went to drug shops, and only 22.6% visited HIV/AIDS care centres. Almost 40% waited more than 24 hours before initiating treatment. Most (60.3%), reported taking recommended artemisinin-based combination treatments (ACT) but a significant minority took only paracetamol (25.6%) or herbal remedies (3.5%). Most (80%) finished their chosen course of treatment; and completion of treatment was significantly associated with the frequency of suspected malaria occurrence (p = 0.03). Most (62.8%) did not take anti-malaria medication while taking antiretroviral treatment (ART) and almost all (87.6%) reported taking an ACT regimen that could potentially interact with Nigeria’s first-line ART regimen. Our findings suggest the need to pay more attention to malaria prevention and control as a crucial element in HIV/AIDS management in this part of Nigeria and other areas where malaria and HIV/AIDS are co-endemic. Also, more research on ART-ACT interactions, better outreach to community-level drug shops and other private sector stakeholders, and clearer guidelines for clinicians and patients on preventing and managing co-infection may be needed. This will require improved collaboration between programmes for both diseases. Malaria and HIV/AIDS overlap geographically in sub-Saharan Africa, Southeast Asia, and South America. Both diseases pose enormous global health challenges with each causing over three million deaths in 2007 and adversely affecting millions more each year , 2. NigeCo-infection with malaria and HIV can make each disease more severe and potentially more infectious. Studies have shown that malaria infection increases plasma HIV viral load even with asymptomatic parasitaemia –9. In tuCo-morbidity also complicates treatment for both diseases as many standard, first-line treatments for malaria and for HIV interact; placing people living with HIV/AIDS (PLWHA) at high risk of harmful adverse events including hepatotoxicity if incorrect antiretroviral drug regimens are prescribed –19. If pConsidering the high rates of co-morbidity and the significant treatment challenges posed by malaria and HIV co-infection, it is important to understand how PLWHA are currently preventing and treating malaria. However, while there is an extensive literature on malaria care-seeking behaviour in Africa –23 thereThis study therefore aims to describe malaria care-seeking behaviours among HIV-infected patients receiving antiretroviral treatment in South-Eastern Nigeria. Our specific research questions are: 1) How common is self-reported malaria co-infection among this sample of PLWHA? 2) To what extent do PLWHA in our sample use informal services (including self-treatment) to diagnose and treat suspected malaria? 3) What medications do PLWHA in our sample use to treat suspected malaria? 4) Do PLWHA alter their antiretroviral treatment practices when taking anti-malarial drugs?It is hoped that the study results will provide information to inform the development and application of effective interventions that would improve the prevention and management of malaria among HIV-infected patients.The study was carried out in Owerri, South Eastern Nigeria, at the Imo State Specialist Hospital (IMSSH) and the Heart-to-Heart unit of the Federal Medical Centre (FMC). The HIV care centres in these facilities are among the major sources of comprehensive HIV treatment and support for PLWHA in South Eastern Nigeria. South-Eastern Nigeria is one of the geopolitical zones in Nigeria, consisting of five states and inhabited by the Igbo-speaking ethnic group. The area covers a landmass of 41,440 km², characterized by tropical rainforest vegetation. Apart from state capitals and a few other urban towns, the area is rural. South-Eastern Nigeria has two distinct seasons, the rainy and the dry seasons. Its climate and environment provide favourable conditions for the breeding of the mosquito vector and malaria endemicity. The area has many socioeconomic characteristics that increase the population-level risk of HIV infections. Malaria co-infection among asymptomatic and symptomatic HIV seropositive individuals in South-Eastern Nigeria has been reported to be 11.8% and 33.3% respectively .The study has a descriptive, cross-sectional design. The study population comprised of all patients living with HIV registered in Imo State Specialist Hospital in Owerri and Federal Medical Centre in Owerri, from October 1 to December 31, 2017, as noted in hospital records . The study sample size was determined using the Taro Yamane method for survey-related studies , to obtaBecause of HIV-related stigma and the tendency for PLWHA to shy away from public activities, we envisaged that a significant proportion of HIV-infected patients would decline to participate in the study. We, therefore, oversampled an additional 20% of the calculated sample size to account for attrition and arrived at a sample size of 409. However, nine of the selected participants did not give consent to participate in the study, leaving us with the final sample size of 398.Systematic sampling was used to select study subjects from both study HIV care centres. We sampled patients over four weeks (two weeks for each centre) during the clinic appointment days for PLWHA. Initially, the number of eligible participants was obtained from the records at each HIV care centre. Only those who were available on the appointment days were selected. On each appointment day, we first divided the number of expected patients who had appointments as listed on the health centre’s records, by the required sample size to obtain an interval number. This interval number was then used to systematically sample patients as they arrived for their appointments on their respective appointment days. For example, if the day’s interval was five, each fifth patient who arrived for an appointment was recruited to participate in the study. In situations when a selected patient declined to consent to participate, the next patient to arrive was selected. The records were crosschecked to ensure that subjects were not selected more than once.The instrument for data collection was a structured pre-tested questionnaire see . The queData were collected using the structured, pre-tested questionnaire by a team of trained research assistants under the supervision of study investigators. To each study participant, the study was first introduced, and the objectives elucidated. Informed verbal consent was also sought and obtained before the questionnaire was administered. Literate respondents self-administered the survey in either English, Igbo, or “Pidgin English”. Research assistants stayed on hand to clarify points of confusion and collected the questionnaires immediately after completion. For patients who could not self-administer the survey either due to illness or lack of literacy, the research assistants verbally administered the questionnaire to them in the local (Igbo) language, English or “Pidgin English” based on the subjects’ choice. The responses were then immediately translated and/or transcribed into English and filled out on the questionnaire by the trained research assistant. Data collection occurred over a one-month period see .p) less than or equal to 5% were considered statistically significant. Analyses were performed on IBM-SPSS Statistics version 23 and Stata version 14.Initial analysis consisted of calculating descriptive statistics to create percentage and frequency tables. Pearson’s chi-squared test (χ2) was performed to examine associations between completing anti-malarial treatments, patterns of reported suspected malaria (re)-occurrence, and having adverse reactions to simultaneous ART and malaria treatment. We also performed multivariate logistic regression to assess associations between socio-demographic factors such as educational attainment, urban residence, and gender and the use of formal or recommended healthcare services for malaria diagnosis and treatment. Probability values  related to their treatment they had experienced. If they selected any of the adverse events from the list, they were considered as having had an adverse event. These six measures were coded as dichotomous outcome variables for our multivariate logistic regression analysis of factors associated with healthcare seeking behaviour.Ethical approval was given by the Ethical Committee of the School of Health Technology, Federal University of Technology Owerri, Nigeria. Permission was also sought and obtained from the management of Imo State Specialist Hospital Owerri and the Federal Medical Centre Owerri, South Eastern Nigeria. Informed verbal consent as permitted by the Ethical Committee was sought and obtained from all the participants before they could take part in the study. Research assistants read informed consent form material to participants and noted the respondents’ consent on the form when given. Verbal consent was obtained instead of written consent because of concerns about patient literacy and protecting patient confidentiality considering the heavy stigma that HIV-infection can carry in this region.There were very few newly HIV-diagnosed patients; only 11% had lived with HIV for less than a year; 48% had been aware of their status for 1–5 years and 38% for 6–10 years see . Almost Few patients sought appropriate, formal care when they suspected that they had malaria. The plurality (39%) used drug shops to buy malaria drugs from shop attendants when they suspected an attack and most others (29%) self-treated at home. Only 23% reported their suspicion to their HIV/AIDS care centres for proper diagnosis see . Most reWhen seeking formal confirmation for suspected malaria an almost equal proportion rely on clinical examination (42%) or laboratory tests . Approximately 12% do not use any form of formal confirmation of diagnosis.Self-treatment was less prevalent among respondents with no formal schooling  than among those who had completed primary, secondary, or tertiary schooling. There was also some indication that men in this sample were less likely to self-treat than women although this association was only significant at the 10% level .Only a small minority of patients received their malaria treatment medications from the formal sector see . More thMost patients reported using recommended malaria treatment medications, with 60% saying that they used ACTs for treatment. However, a significant minority 26%) only used antipyretics  and other almost 10% used a non-ACT anti-malaria medication. Use of traditional medicines was low, with only 3.5% saying that they used herbs. The most common ACT regimens used were artemether/lumefantrine (60%) which is the treatment recommended by the Nigerian Ministry of Health, [% only usApproximately 80% of patients said that they usually complete their chosen malaria treatment course. Those who reported very frequent (monthly) or very infrequent  suspected malaria occurrences were significantly more likely to complete their malaria treatments (p = 0.03) than those who reported quarterly occurrences see . We founWe found that 62.8% of respondents said that they did not take their chosen anti-malaria drugs while they were taking their antiretroviral drugs. Respondents who took herbs or alternative drugs had significantly higher odds of continuing ART while taking malaria treatment than those taking ACTs. The adjusted odds of not stopping ART were 6.14  for patients who took herbs to treat their malaria and 3.94  for patients who took non-ACT malaria treatments See . Respond2 = 24.5) with 22.5% of those who self-report having monthly bouts of suspected malaria reporting adverse events, 14.0% of those with reported quarterly attacks, and 18.8% of those reporting annual attacks respectively  were more likely to report adverse events than rural and respondents in the middle age groups than the youngest age group  see . Those wAlmost none of the respondents (87%) could provide a recommendation for ways to improve malaria prevention and treatment for PLWHA. The few suggested actions that were offered were combining malaria treatment with HIV drugs (1%), suspending antiretroviral drugs while on malaria treatment (7%), and using treated mosquito nets to prevent further mosquito bites (4%) see .This study examined malaria care-seeking behaviour among HIV/AIDS patients receiving antiretroviral therapy in Southeastern Nigeria. We found extremely high levels of self-reported co-infection with all patients reporting having had suspected malaria and almost 80% reporting a suspected re-occurrence on at least a quarterly basis. Our data suggest that most of these suspected cases are never formally diagnosed but rather assumed to be malaria and treated as such. This highlights the need for greater access to rapid diagnostic tests, which were only used by 16.1% of our sample.The rate of malaria co-infection reported by our study participants is higher than that found in other sub-Saharan African settings , which iWe found heavy use of informal sources such as drug shop sellers and home-care for immediate treatment on suspicion of malaria. Self-medication and use of informal drug sellers are well documented among the general population in Nigeria , 29 and What is surprising is that this study sample, which has steady access to relatively well-resourced health facilities, has treatment patterns like those found in the general public. This highlights the importance of drug cost , and of convenience factors in driving healthcare-seeking behaviour. The finding that self-treatment was more common among respondents in our study with higher levels of education lends additional support to the argument that convenience and quality of care are important drivers of self-treatment.Our findings regarding the heavy use of drug sellers and private laboratories also underscore the continued importance of engaging the private sector in HIV/AIDS and malaria program development and of pursuing community-based treatment strategies to improve access to appropriate treatment. Our study and others indicate that there is a broad range of healthcare stakeholders in both the public and private sectors that are involved in malaria control among HIV-infected patients, including hospital clinics, pharmacies, private clinics, and patent medicine stores. For example, a recent Nigerian study reported that the private sector took care of more than 43% of malaria cases .We find that the majority of patients using anti-malaria medications (as opposed to drugs for symptom relief) reported using the first-line, recommended ACTs. There were few reports of mono-therapy or of ineffective treatment, and approximately 80% of patients said that they finished their course of treatment. Although the quality of these medications is unknown as many were purchased privately, it is still heartening to find that newer, recommended treatment regimens are widely available and being taken. We must note, however, that a significant minority of respondents took only paracetamol, sub-optimal anti-malarial treatments, or herbal remedies.We note two areas of concern regarding HIV and malaria co-treatment. First, many patients report using ACT regimens containing drugs  that interact with the antiretroviral drugs included in Nigeria’s first line ART regime . A significant minority of study patients combine anti-malaria drugs with ART, suggesting a risk for adverse events. In addition, patients were more likely to continue ART when they were taking non-ACT anti-malaria medication or herbal remedies (which may explain why less educated patients were more likely to report continuing ART than their more educated counterparts).The association between continuing ART and using traditional or non-ACT medications may be due to patients experiencing fewer drug interactions when taking these alternative medications or to them perceiving alternative regimens as relatively safer or milder and, therefore, being less afraid of remaining on ART while taking them. Other studies in sub-Saharan Africa have found that HIV-infected patients do concurrently use herbal treatment while on ART for perceived minor ailments  in the belief that it increases their energy and immunity . AlthougAnother concerning finding was that 80% of patients stopped taking ART when they started malaria treatment—a behaviour that increases the risk of resistance to antiretroviral medications and leads to poor viral load suppression. Moreover, the responses (or lack thereof) to the question asking for suggestions about to improve care, suggest that patients did not know the proper course of action for managing co-infection. Given the complexities of malaria treatment in patients receiving ART and the extremely high self-reported malaria incidence, more effort must be made to strengthen and develop new malaria prevention interventions and to better integrate them into HIV/AIDS care. Effective prevention interventions exist. For example, we know that co-infection is significantly lower among patients who use insecticide-treated bed nets and co-trimoxazole chemotherapy , 37. EveA surprising finding is that most of the subjects studied knew the names of the drugs they use to treat malaria. This contrasts with findings in a recent Nigerian study , in whicThe finding that almost no study respondents could give suggestions for managing or preventing co-infection suggests that the structure of counselling sessions may not provide enough time to discuss such topics and provide updates. More comprehensive counselling sessions using techniques such as question and answer sessions and video recordings might help to fill gaps in patient knowledge. Providers and HIV clinic management staff should also develop systems to more closely track which malaria drugs are being used by patients.This study is limited by its narrow geographic scope and modest sample size and non-random sample selection strategy, which reduces its generalizability. The study is subject to both recall and social desirability bias as respondents may not remember past malaria episodes or treatment behaviour accurately and may report the health-seeking behaviour that they perceive as desirable. Problems with recall may be exacerbated by the fact that patients with a serious, life-threatening illness may be under great physical and emotional stress, which may impair memory. We attempted to address recall bias by having data collectors on-hand to reword or repeat questions, and prompt memory but this, in turn, may have introduced measurement error if questions were explained differently by different data collectors. The lack of back-translation of our survey instrument may have also introduced measurement error. Social desirability bias, if it occurred would have most likely lead to an under-reporting of the significant levels of informal care reported by participants. Finally, as noted at the start of this discussion, our estimates of malaria occurrence are based on self-report rather than diagnosis and, therefore, not precise. While this limits our ability to measure the level of co-infection in this population it does not greatly limit our ability to describe the care-seeking behaviour of these HIV-infected patients when they suspected that they have malaria.This study has shown that PLWHA receiving ART in our sample self-report extremely high levels of suspected malaria, and that very little of their subsequent malaria care occurs in the formal sector. More worryingly, frequent bouts of suspected malaria seem to lead to interruptions in ART as most patients do not take ART and anti-malaria drugs simultaneously. Patients who do continue ART while treating malaria often use alternative, non-ACT anti-malaria drugs or herbal remedies, neither of which would tend to have well documented drug-drug interaction profiles with antiretroviral drugs. Our findings suggest the need for more research on ART-ACT interactions; better outreach to community-level drug shops and other private-sector stakeholders; and clearer guidelines for clinicians and patients on monitoring and managing co-infection. Wider access to rapid malaria diagnostic tests might also reduce the high level of presumptive malaria self-treatment that we find. Providing anti-malaria drugs for free in HIV care programs may also be worth exploring. Overall, we suggest that malaria prevention and control be viewed as an important element in the treatment HIV/AIDS patients in malaria-endemic countries such as Nigeria. This will require improved collaboration between programs for both diseases.S1 Appendix(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Vagus nerve stimulation (VNS) exerts a cortical modulating effect through its diffuse projections, especially involving cerebral structures related to autonomic regulation. The influence of VNS on cardiovascular autonomic function in drug-resistant epilepsy patients is still debated. We aimed to evaluate the impact of VNS on cardiovascular autonomic function in drug-resistant epilepsy patients, after three months of neurostimulation, using the heart rate variability (HRV) analysis.Multiple Trigonometric Regressive Spectral analysis enables a precise assessment of the autonomic control on the heart rate. We evaluated time and frequency-domain HRV parameters in resting condition and during sympathetic and parasympathetic activation tests in five epilepsy patients who underwent VNS procedure.We found appropriate cardiac autonomic responses to sympathetic and parasympathetic activation tests, described by RMSSD, pNN50, HF and LF/HF dynamics after three months of VNS. ON period of the neurostimulation may generate a transient vagal activation reflected on heart rate and RMSSD values, as observed in one of our cases.VNS therapy in epilepsy patients seems not to disrupt the cardiac autonomic function. HRV represents a useful tool in evaluating autonomic activity. More extensive studies are needed to further explore cardiac autonomic response after neurostimulation. Epilepsy affects approximately 65 million people worldwide . AlthougPatients with drug-resistant epilepsy, defined as seizure occurrence despite judicious association of two or more antiepileptic drugs over a minimum of 12 months period , may benVNS exerts through its diffuse projection via the nucleus of the solitary tract, or the reticular system, a cortical modulating effect, especially involving cerebral structures related to autonomic regulation, such as thalamus, amygdala or prefrontal region . The impHeart rate variability (HRV) represents a simple and non-invasive method to evaluate the sympathovagal balance, outlining the cardiac ability to adapt to hemodynamic and pathological conditions. Sympathetic hyperactivation and reduced cardiac vagal modulation associated with low HRV determines higher risk of cardiac arrhythmia and sudden death .Rüdiger et al. proposed a novel algorithm to detect physiological oscillations of the heart rate (HR) based on RR intervals measurements – multiple trigonometric regressive spectral (MTRS) analysis . HRV anaThe aim of this research is to evaluate the impact of the VNS on cardiovascular autonomic function, through sympathetic and parasympathetic activation tests, after three months of neurostimulation in drug-resistant epilepsy patients, using MTRS analysis of the HRV.ECG recordings of the first five drug-resistant epilepsy patients from our department who underwent left laterocervical VNS procedures were analyzed using MTRS software.None of the five patients underwent epilepsy surgery and had no cardiovascular comorbidities, as well as no cardiovascular medication. No changes in the antiepileptic medication were performed three months prior to our first evaluation and between the two HRV tests.ECG was registered before the VNS procedure and at least three months after theimplantation of the electrode. ECG recordings were performed during ON and OFF time period of VNS stimulation.Blood tests including electrolytes, hepatic and renal function were within the normal range for all patients in both evaluations.All patients were informed about the study protocol and gave written consent in accordance with ethical principles. The study was carried out in accordance with the Helsinki Declaration.We applied a standardized protocol including resting state and subsequently four autonomic activation tests, each test entailing a five minute ECG recording. There was a HR stabilization before starting the evaluation. Tests were performed at the same time range (4-6 PM), after 30 minutes of resting position in clinostatism, at a constant temperature of 22ᴼC, in a quiet room, in the absence of sounds or human voices, without prior physical effort or ingestion of caffeinated or alcoholic beverages 24 hours before the evaluation. All patients were asked to empty the bladder before starting the evaluation. Moreover, the tests were performed at least three hours after lunch in order to avoid gastric distension. Two sympathetic activation tests were performed: a maximal voluntary isometric contraction of the fist, using a dynamometer – “hand-grip” test and a three-minute standing test. Two parasympathetic activation tests were considered: “deep breathing” test, consisting of six complete cycles of deep inhale and exhale over 60 seconds, with timing, 10 seconds for each cycle, and Valsalva maneuver. The test sequence was standardized: resting state, deep breathing test, hand-grip test, standing test and Valsalva maneuver for all patients.® acquisition system was used for data collection and analysis. BIOPAC® represents an integrated hardware and software system that converts biologic parameters like HR to numeric data. AcqKnowledge software version 3.9.1.6 was used for refining the recorded data. Data processing was done using MTRS software version 7.3.2.0 . Before each analysis, a manual data correction of ECG artifacts was carried out.BIOPACThe dynamic assessment of HRV by MTRS analysis allows a precise evaluation of cardiovascular modulation under different conditions. This software assesses the HRV time-domain and frequency-domain parameters, based on the trigonometric regressive analysis . The HRVMTRS works with two data segments, a global and a local data segment. The global data segment considered was the length of the ECG recording (5 minutes) and the local data segment was set - 30 seconds. The analysis is performed in all local data segments, until the end of the global data segment. All oscillations of the biosignals  are analyzed using a trigonometric function of the parameters A (amplitude), ω (frequency), and ϕ (phase shift) . For eacHeart rhythm oscillations may be categorized into four primary frequency bands: Ultra Low Frequency (ULF), Very Low Frequency (VLF), Low Frequency (LF) and High Frequency (HF). Respiration modulates vagal activity and contributes to the HF component of the spectra, ranging from 0.15Hz to 0.4Hz . Recent The following time-domain parameters were considered: pNN50  and RMSSD . These two parameters reflect the parasympathetic influence on the cardiac rhythm .HRV is significantly correlated with an average HR, dependent on the influence of autonomic nervous system activity  and alsoThis correction procedure does not remove any information about the signal’s oscillations but only makes the oscillations relative to the signal’s average value . This alHRV is also influenced by the respiratory rate (RespRate). Therefore, alterations in these parameters may impose changes in HRV , 21. A dThe analysis was performed using GraphPad Prism software version 8.1.0. For the statistical analysis of the data, having into consideration the small sample size, series normalization was very difficult. Wilcoxon matched-pairs tests were applied to compare the parameters of the analyzed series. Spearman’s rank correlation coefficient (rs) was used to assess the relationship between variables, and Student’s t-test or non-parametric Mann–Whitney test were employed to determine differences. The significance level  was considered to be 0.05 (5%).Clinical symptoms, type of epilepsy, age of onset, cerebral MRI findings and current treatment are depicted in Seizure frequency decreased for patient 3 in the last month before the second HRV evaluation, during VNS therapy. VNS parameters of the five patients are summarized in Our ECG recordings did not reveal bradycardia, tachycardia or other cardiac arrhythmias in resting state or during challenge for all five patients. MTRS analysis of the ECG recordings from the five patients during resting state and autonomic activation tests provided time-domain  and freqIn order to analyze the relationship before and after correction of the parameters, we performed Wilcoxon matched-pairs signed rank test. Spearman’s rank correlation coefficient “rs”, calculated comparing values before and after normalization, is displayed in The HR and RespRate correlation with the same above mentioned parameters is displayed in The response pattern to autonomic activation tests  displayed by several HRV parameters was similar after normalization  and respectively sympathetic (hand-grip and standing) activation tests were observed for all patients, indicated by increases in RMSSD, pNN50 and HF values, respectively decreases of the values of the aforementioned parameters during challenge, in both evaluations.The first patient presented a slight decrease of RMSSD values after three months of VNS, while the second, the third and the fifth patient displayed an increase of several parasympathetic specific parameters values in the second test. Time and frequency-domain parameters did not reveal a significant change in the cardiac autonomic state after three months of VNS for the fourth patient .The frequency-domain parameters for patient 3 during the second HRV evaluation in resting state are illustrated in Patients with refractory epilepsy may present decreased HRV, raising the concern that altered autonomic function might contribute to sudden unexpected death in epilepsy (SUDEP) , 26. LonThe positive effect of VNS in patients with drug-resistant epilepsy is considered to be mediated by the afferent pathways of the vagus nerve, modulating the activity of different cerebral structures, probably involved as trigger-points of seizures . ActivatAll five patients presented normal responses to sympathetic and parasympathetic activation tests in the first HRV evaluation. VNS did not alter HR modulation in response to autonomic activation tests after three months of vagal neuromodulation. This data may indicate the minor contribution to cardiac control of the sympathetic efferent axons contained within the vagosympathetic complex in response to VNS. Cervical vagus nerve is a mixed nerve containing both afferent and efferent axons, belonging to the sympathetic and parasympathetic nervous system , 33. Carpredominantly parasympathetic preganglionic axons . ParasymChronic VNS influences both sympathetic and parasympathetic modulation but does not negatively influence autonomic cardiovascular regulation , 40. Incactivity seems to have cardioprotective effects against SUDEP . CardiacAnother cortical structure involved in autonomic regulation is insula. Insular lesions are associated with mortality through autonomic dysfunction . ElectriAntiepileptic drugs can also influence autonomic activity. Carbamazepine has been shown to affect the autonomic modulationamong patients with temporal lobe epilepsy , 54, andThe strengths of our report include MTRS analysis of the HRV on short ECG recordings (5 minutes) after autonomic activation tests. The advantage of MTRS analysis, which does not need interpolation on non-equidistant RR intervals contrary to Fast Fourier Transformation, is the assessment of shorter local data segments , 13. An One of the limits of our report is the reduced number of patients while the other is using surface EEG study, because of the distant location of the insular cortex relative to scalp electrodes and the rapidly spreading activity.Autonomic nervous system should be evaluated in epilepsy patients because of the risk of cardiac arrhythmias and SUDEP, particularly in the drug-resistant ones. We propose HRV analysis as a useful tool to assess sympathovagal balance and identify high-risk patients for cardiac arrhythmias. Moreover, HRV analysis could be a practical tool in identifying suitable patients for VNS therapy.


T1 and T2 weighted images. From these findings, CPPD crystal deposition in the ligamentum flavum was suspected preoperatively. Biopsy at the level of C5-6 were performed to confirm the diagnosis. Perioperative and histopathological findings revealed that CPPD crystals were deposited along the dorsal dura mater, not in the ligamentum flavum. We firstly report the CT and MR imaging features of a possible new concept in the differential diagnosis of CPPD crystal deposition disease.Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is characterized by the accumulation of CPPD crystal in articular and periarticular tissues, but CPPD crystal deposition along the spinal dura mater has not been previously reported. We report a 54-year-old male presenting with progressive neck pain and numbness of the extremities. CT showed diffuse dorsal epidural calcification from C3-T6 which resulted in spinal canal stenosis. On MR imaging, the lesion was hypointense on both  The disease can also be asymptomatic.4 The most commonly affected joints are knees, followed by wrists, hands, pelvis, symphysis pubis and hips. Although CPPD crystal deposition disease of the spine is less common, it can involve the ligamentum flavum; longitudinal, supraspinous and interspinous ligaments; the intervertebral discs, and the sacroiliac and apophyseal joints.2 However, CPPD crystal deposition along the dorsal spinal dura mater has not been previously reported. We herein describe a case of diffuse calcification in the posterior epidural space at C3-T6 mimicking previously reported CPPD crystal deposition in the ligamentum flavum.5 Pathological examination after biopsy proved the lesion to be CPPD crystal deposition along the dura mater.Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is characterized by the accumulation of CPPD crystals in articular and periarticular tissues.–1; normal, 10–40 mg dl–1). Unenhanced CT of the cervicothoracic spine showed diffuse dorsal epidural calcification at C3-T6. The lesion presented as a crescent shape along the spinal dura mater and had a well-defined border along the vertebral arch , and there was no evidence of malignancy. These features confirmed the diagnosis of CPPD crystal deposition disease along the spinal dura mater. The patient’s neurological symptoms during the postoperative course was uneventful, and follow-up CT performed 4 months later revealed no regrowth of the calcified lesion.A 54-year-old male presented with progressively worsening neck pain and numbness of the left upper extremity and the left face for 2 weeks. He had no history of recent head or neck injuries. He was followed closely without further treatment because of his fluctuating subjective symptoms. However, the symptoms gradually worsened, and he visited our hospital for intensive examination and treatment 2 months after the onset. Neurological examination revealed a mildly spastic gait and exaggerated deep tendon reflexes in the bilateral upper and lower extremities. He also had mild dysuria and constipation. Muscle strength and vibration sense were normal, and no Babinski sign was present. Peripheral blood examination on admission revealed no significant abnormality. Cerebrospinal fluid examination was within normal limits except for slightly elevated protein , ossification of the ligamentum flavum, amyloidosis and hyperparathyroidism associated with chronic renal failure or alone and tuberculosis. CLF predominantly affects elderly women and frequently occurs in the lower part of the cervical spine.In conclusion, we believe we are the first to report CPPD crystal deposition disease along the spinal dura mater. We observed a multilevel, crescent-shaped, diffusely calcified lesion with a well-defined border with the vertebral arch, which may be characteristic of CPPD crystal deposition along the dorsal spinal dura mater, rather than in the ligamentum flavum.This is the first report describing a patient with calcium pyrophosphate dihydrate (CPPD) crystal deposition along spinal dura mater.Preoperatively, CPPD crystal deposition disease in ligamentum flavum was suspected.CT showed multilevel, crescent-shaped, diffusely calcified lesion with well-defined border with the vertebral arch, which may be characteristic of CPPD crystal deposition along the dorsal spinal dura mater, rather than in the ligamentum flavum.Written informed consent for the case to be published  was obtained from the patient(s) for publication of this case report, including accompanying images. This work was supported by Grant-in-Aid for Scientific Research (C) Grant Number 17K10365.


Sinus of Valsalva aneurysm (SVA) is an uncommon cardiac anomaly, with an incidence of less than 1% of open heart surgery cases. Its evolution is most frequently silent, being found incidentally or discovered in the event of its acute rupture. Non-ruptured giant SVAs may cause unusual clinical manifestations, as a consequence of their protrusion into the heart chambers or compression of the coronary vessels and are frequently associated with aortic insufficiency of various degrees of severity. The gold standard treatment for SVAs consists of complete replacement of the aortic root and valve. However, in certain cases, valve-sparing procedures may prove to be a more suitable alternative.A 68-year-old male patient presented with dyspnea as symptom caused by a large (> 5 cm) right sinus of Valsalva aneurysm. The aneurysm was occupying most of the right ventricle and was associated with severe aortic regurgitation. The surgical treatment of the condition involved valve-sparing root reconstruction procedure (remodeling technique), completed with external stabilization of the aortic valve annulus via running suture annuloplasty. Following the uneventful intervention, the patient did well and his status improved. The follow-up transthoracic echocardiography obtained 1 month after surgery showed a fully competent aortic valve with no regurgitation.Despite complete aortic root and valve replacement being considered the safest approach to large SVAs complicated with aortic insufficiency, valve-sparing procedures should not be overlooked in case of a dilated aortic root with uncalcified aortic valve. Performing valve-sparing by applying a remodeling technique operation completed with annuloplasty reduces aortic valve insufficiency, avoiding side-effects related to implanted valves. Sinus of Valsalva Aneurysm (SVA) is considered to be an uncommon cardiac anomaly, being found in less than 1% of the cases in cardiac surgery . SVA mosThe typical evolution of SVAs is towards rupturing, thus carrying a very high rate of mortality. The likelihood of a fatal episode occurring has been shown to be correlated with maximum aneurysm size . HoweverIn selected cases involving small SVAs, a primary closure or patch closure of the aneurysm may be performed . HoweverA 68-year-old male patient with no significant past medical history was admitted because of progressive shortness of breath (NYHA III-IV) which began 6 months prior to the admission. Transthoracic echocardiography (TTE) revealed an aneurysmal bulge of the right aortic sinus into the right ventricle without obvious rupture. The patient had smoked for more than 50 years, denied any alcohol consumption and took no medication.On TTE, an extensive unruptured SVA was visible, arising from the right coronary sinus of Valsalva and protruding into the right ventricular chamber, significantly comprising the cavity. TTE also revealed prolapse of the right coronary cusp of the aortic valve and severe (grade IV) aortic valve regurgitation on an otherwise uncalcified native tricuspid aortic valve. The left ventricular systolic function was depressed, with an ejection fraction of 40%, presumably as a consequence of the long-standing progression of the aortic valve regurgitation . TTE revealed no other valvular dysfunctions.Computed tomography angiography (CTA) confirmed the presence of a saccular aneurysmal structure originating from the right coronary sinus of Valsalva, protruding into the right ventricular chamber, measuring 50,2 mm × 50,9 mm × 64 mm Fig. a. The diAs part of the preoperative preparation, because of the depressed LV function, levosimendan was administered. The surgical intervention was performed under general anesthesia. After median sternotomy, cardiopulmonary bypass was applied by cannulating the ascending aorta and the right atrium. Myocardial preservation was obtained via cold Custodiol crystalloid cardioplegia. The aortic valve was exposed using a transverse aortotomy. The intraoperative discoveries confirmed the above findings: a giant aneurysm originating from the right aortic sinus was protruding into the right ventricle Fig. a, b, whiThe patient’s recovery was favourable. The follow-up TTE obtained 1 month after surgery showed a fully competent aortic valve with no regurgitation.Sinus of Valsalva aneurysm is defined as an abnormal dilatation of the aortic root situated between the aortic valve annulus and the sinotubular junction. Most of these aneurysms originate from the right sinus of Valsalva and are usually isolated. SVAs may be congenital in origin, caused by the weakness of the elastic and muscular tissues of the aortic wall, as encountered in patients suffering from hereditary connective tissue disorders, such as Marfan or Ehlers-Danlos syndrome , 19. LesSVAs are highly uncommon, often presenting with silent evolution or unspecific symptoms, such as angina-like chest pain, palpitations, or dyspnea, as in the case presented above. They are frequently detected only as incidental findings or in the event of acute rupture, their most severe and potentially fatal complication. Several cases have been reported in which SVAs caused unusual cardiac manifestations, such as ischemia, conduction disturbances, or right ventricular outflow tract obstruction.Besides conventional angiography, other noninvasive imaging modalities may prove useful in assessing SVA characteristics, such as TTE, cardiac CTA Fig. , and carLevosimendan has been shown to have a beneficial effect on cardiac function, causing fewer side effects than catecholamines. In cardiac surgery, postoperative low cardiac output syndrome (LCOS) may result in multiple organ failure and high mortality. Preoperative administration of levosimendan is recommended in patients with depressed left ventricular function undergoing cardiac surgery because it improves hemodynamics and optimizes postoperative organ function, lowering the risk of LCOS .Regardless of etiology and clinical expression, it is generally agreed that SVAs should undergo surgical correction, despite the surgical treatment modality being a matter of controversy . The safEven in the case of large unruptured SVAs accompanied by severe aortic valve insufficiency, valve-sparing procedures may prove to be feasible. The age and functional status of the patient, as well as the possibility to avoid implanted valve side effects, may prompt repair strategy. Published data about cases of SVAs surgically managed by valve-sparing techniques is scarce. More than that, a giant SVA associated with severe aortic insufficiency surgically managed with a modified suture annuloplasty technique has not been previously reported. Therefore, we present this case hoping that it will encourage surgeons to consider valve-sparing techniques as a real possibility of treatment in such cases.


Kinematic gait analysis is an important noninvasive technique used for quantitative evaluation and description of locomotion and other movements in healthy and injured populations. Three dimensional (3D) kinematic analysis offers additional outcome measures including internal-external rotation not characterized using sagittal plane (2D) analysis techniques.The objectives of this study were to 1) develop and evaluate a 3D hind limb multiplane kinematic model for gait analysis in cats using joint coordinate systems, 2) implement and compare two 3D stifle (knee) prediction techniques, and 3) compare flexion-extension determined using the multiplane model to a sagittal plane model. Walking gait was recorded in 3 female adult cats . Kinematic outcomes included flexion-extension, internal-external rotation, and abduction-adduction of the hip, stifle, and tarsal (ankle) joints.in vivo were similar to joint angles determined using a feline hind limb skeleton in which markers were placed directly on landmarks ex vivo. Differences in hip, stifle, and tarsal joint flexion-extension were demonstrated when comparing the multiplane model to the sagittal plane model.Each multiplane stifle prediction technique yielded similar findings. Joint angles determined using markers placed on skin above bony landmarks This multiplane cat kinematic model can predict joint rotational kinematics as a tool that can quantify frontal, transverse, and sagittal plane motion. This model has multiple advantages given its ability to characterize joint internal-external rotation and abduction-adduction. A further, important benefit is greater accuracy in representing joint flexion-extension movements. Kinematic gait analysis is an important noninvasive technique that is used to quantify locomotion and other movements in healthy and injured populations –3. Most Gait analysis using multiplane kinematics allows for quantification of joint angles in the sagittal, transverse, and frontal planes to describe flexion-extension, internal-external rotation, and abduction-adduction –17. The To our knowledge no study has investigated sagittal, frontal, and transverse plane kinematics of walking gait in the cat, and a robust pelvis and hind limb kinematic model has not been described. The purpose of this study was to 1) develop and evaluate a 3D multiplane kinematic model of the cat pelvis and hind limbs for gait analysis using anatomically-oriented joint coordinate systems, 2) implement and compare two 3D stifle prediction techniques, and 3) compare flexion-extension determined using the 3D multiplane model to a sagittal plane model.th metatarsal, and mediodistal aspect of the 2nd metatarsal. An additional marker was placed bilaterally approximately 2.5 cm proximal to the lateral malleolus on the lateral aspect of the fibula. Three representative overground gait trials with a minimum of 3 consecutive step cycles each at consistent speeds were selected for each subject. Each step cycle was resampled into percent step cycle and divided into stance and swing phases. Gaps in data < 5 frames were filled using Woltring quintic splines. The fourth cat was included for model validation, was not a subject during in vivo data collection, and was euthanized with an overdose of sodium pentobarbital (>50mg/kg) and transcardial perfusion with saline for reasons unrelated to this study. For use during model validation, the pelvis and hind limbs were disarticulated at the sacrum, and all soft tissues were excised except hip, stifle, and tarsal joint connective tissues  which were left intact. The specimen initially was exposed to a fixative  and then maintained in water at 4°C to prevent specimen deterioration and stiffening of the connective tissues.All procedures were approved by the University of Louisville and Robley Rex VA Institutional Animal Care and Use Committees (IACUC #12113). Four purpose-bred specific-pathogen free spayed female adult cats were included in this study. Three cats were conditioned to cross a 4.6 m walkway (0.36 m width) at their self-selected walking speeds approximately 30 times daily, 5 times per week for food rewards. Motion capture data were acquired using a 3D test space encompassing the entire width and length of the walkway . Data weDue to skin motion artifact across the stifle joint, a retroreflective marker was not placed on the cat stifle. Instead, two techniques were used to determine a lateral stifle virtual marker. For the first technique (unadjusted tibia axis technique), the lateral stifle virtual marker was defined as the midpoint of the line connecting 1) the vector of length equal to the fibula length originating at the lateral malleolus projected through the vector marker and 2) the vector of length equal to the femur length originating at the greater trochanter projected towards the fibula vector endpoint . For theAnatomical segment coordinate systems were developed for the pelvis and bilateral femur, tibia, and tarsus based on markers placed on the skin . Each anRotations of the distal segment relative to the proximal segment about three clinical axes were determined using joint coordinate systems , 17. Fleth metatarsal markers. Kinematic outcomes were verified using synched video analysis.Data were analyzed using motion analysis software  and a custom script . Three-dimensional kinematics were determined for the hip, stifle, and tarsal joints of each hind limb. Kinematic angles in the sagittal (flexion-extension), transverse , and frontal (abduction-adduction) planes were determined. Stifle kinematic analysis was conducted for each stifle prediction technique. Flexion-extension determined using the multiplane kinematic model was compared to flexion-extension determined using a 3 coordinate sagittal plane kinematic model . For theThe multiplane kinematic model was applied to the right hind limb of the cadaveric specimen. During testing the specimen pelvis was rigidly fixed using two clamps attached to the right iliac wing and ischial spine at the obturator foramen. The hip, stifle, and tarsal joints could move freely within the constraints of the connective tissues. Markers were adhered directly to bony locations previously described for placement of skin markers. Additionally, a marker triad representing bone oriented coordinate systems was adhered to each hind limb segment  . Each trThe hind limb specimen was manually moved through a walking gait motion . Hip, stifle, and tarsal joints were manipulated collectively in 3D space using a rigid wooden extension attached to the metatarsus. Thin metal wire was threaded through the metatarsus connective tissue and secured to an eyebolt affixed to the rigid extension. The rigid extension could rotate relative to the tarsus while providing translational constraint. Nine cycles were recorded, and data were normalized by resampling into percent cycle.3 is the rotation matrix from the proximal segment bony landmark coordinate system to the proximal segment marker triad coordinate system, R2 is the rotation matrix from the distal segment bony landmark coordinate system to the proximal segment bony landmark coordinate system, and R1 is the rotation matrix from the distal segment marker triad coordinate system to the distal segment bony landmark coordinate system. The rotation matrices R1, R2, and R3, are each notationally equivalent to1, θ2, and θ3 are the three rotations to transform one coordinate system into another coordinate system [1 and R3 were determined for one frame with the hind limb mounted in a neutral position  and then applied to all frames. R2 corresponds to the joint angles determined at each frame using the bony landmark coordinate systems of the proximal and distal segments. Rtransform joint angles were compared to joint angles determined using the marker triad coordinate systems of the proximal and distal segments.The joint coordinate system was used to determine 3D joint angles using the marker triads on each segment. Joint angles determined using the bony landmark marker set and the marker triads were compared. The bony landmark marker set and the marker triad marker set defined coordinate system orientations on each segment. Therefore, small differences in marker placement and coordinate system alignment were present and unavoidable. Offset in coordinate system orientations defined by the bony landmark marker set and the marker triad on each segment due to alignment differences between the marker sets was eliminated using coordinate transformation. The bony landmark coordinate system joint angles were related to the marker triad coordinate system joint angles using the following transformation at each joint:e system . R1 and Ages were 2.9 years, mean weight was 3.5 ± 0.2 kg, and mean walking speed was 0.73 ± 0.08 m/s for the three cats performing gait trials. Age was 1.4 years and weight was 3.4 kg for the cat used for model validation.Multiplane stifle kinematics determined using both prediction techniques were similar, and a representative hind limb is shown in Bilateral 3D kinematics for the hip, stifle, and tarsal joints during stance and swing were determined, and a representative hind limb is shown in Ex vivo walking task hip, stifle, and tarsal kinematics were similar using the triad marker set and the transformed bony landmark marker set  could be achieved for scenarios involving object negotiation , slope wIn the current study, hind limb kinematics trends were similar across healthy cats. It is important to note that the magnitudes of the kinematic outcomes, especially internal-external rotation and abduction-adduction, are dependent on the relative orientation of the defined anatomical coordinate systems based on segment motion capture markers applied to each cat. Therefore, subtle differences in defining the mediolateral and/or craniocaudal axes in each segment defining a joint may lead to kinematic outcomes that appear skewed. For instance limb internal-external rotation of the hip varied from 4.8° to 17.7° during gait for the cat reported in Optical motion capture systems allow flexibility to assess gait across a range of tasks. However, it is known that skin motion around the stifle joint in cats and rats prevents reliable stifle tracking using skin-mounted markers and may lead to positional errors which could significantly affect kinematic calculations and lead to overestimation or underestimation of joint angles , 33. FurIn the current study, two stifle projection techniques were implemented and compared. In both techniques, the tibial mediolateral direction was defined based on the medial and lateral malleoli markers. A femoral mediolateral direction based on medial and lateral femoral condyle markers as done in canines , 20 and The sagittal plane and multiplane kinematic models predicted similar gait patterns within a single plane  which isHip joint flexion-extension was consistently larger using the sagittal plane kinematic model compared to the multiplane kinematic model. This difference was not consistently present in a study evaluating dogs  and coulStifle flexion-extension was similar for the sagittal plane kinematic model and the multiplane kinematic model using both stifle projection techniques. These findings are consistent with findings reported for dogs . This stth metatarsal and lateral malleolus markers. If the tarsal joint behaved as a hinge joint with the axis of rotation acting through the lateral malleolus, the lateral malleolus and calcaneus markers would both define the tarsus segment. However, the multiplane kinematic model predicted tarsal joint internal-external rotation and abduction-adduction indicating that the tarsal joint has more degrees of freedom than a hinge joint, which is consistent with tarsal joint passive range of motion measured in cats [Tarsal joint flexion-extension was consistently larger using the sagittal plane kinematic model compared to the multiplane kinematic model. This difference was not consistently present in a study evaluating dogs  and coul in cats . TherefoComparative data are reported for healthy cats in this study, but differences may be more prominent when comparing sagittal plane and multiplane kinematics in animals with abnormal gait patterns due to some sort of injury or developmental abnormality. Internal-external limb rotation may increase in pathologic gait , 39, andex vivo. Joint angle patterns were similar across techniques but differed in magnitude due to slight offsets in orientation of the segment coordinate systems. After adjusting for the difference in orientation for each segment with the limb in a neutral position, measured joint angles differed between the two techniques by < 5° for all joints. These findings further support the use of stifle marker prediction techniques to define stifle kinematics in cats because differences between the bony landmark and marker triad coordinate systems were primarily due to differences in coordinate system setup alignment and not the use of a stifle marker prediction technique. External-internal rotation and abduction-adduction stifle and tarsal joint angles in the cadaver specimen were generally less in magnitude compared to in vivo joint angles. This difference may be due to motion of the hand not replicating actual gait that may have limited movements not contained within the sagittal plane. However, the movements applied to the cadaveric specimen were sufficient for testing and to show applicability of the multiplane model to determine flexion-extension, external-internal rotation, and abduction-adduction.Joint angles determined using the 3D kinematic model based on markers placed on skin overlying bony landmarks were compared to joint angles determined using bone-oriented coordinate systems Overground walking is a daily activity in animals, and is therefore an important movement to evaluate in order to understand neurologic and biomechanical functions. Thus, these first assessments using a multiplane model were conducted on overground walking in healthy cats. Hind limb range of motion for other tasks or in injured cats may exceed the range of motion evaluated in this study. For instance, cats with spinal cord injury traversing pegs with predetermined spacing and contact area demonstrated greater hip flexion and knee extension when adopting a novel placing strategy in which a hind limb crosses the midline to place onto a contralaterally positioned peg . FurtherThe cat multiplane model developed in this study was used to determine flexion-extension, internal-external rotation, and abduction-adduction of the hip, stifle, and tarsal joints. The multiplane model brings greater interpretive power to gait assessment than sagittal plane assessment as it characterizes joint internal-external rotation and abduction-adduction in addition to flexion-extension. Three dimensional stifle kinematics were similar regardless of projection technique used, and although waveforms were similar, the absolute flexion-extension angles may differ when comparing the multiplane model to the sagittal plane model.


Odocoileus hemionus) samples obtained along a 241-km migratory route in western Wyoming, USA, during spring 2015 and 2016. In support of a fitness benefit hypothesis, individuals occupying areas closer to peak forage quality had decreased FGM levels. Specifically, for every 10-day interval closer to peak forage quality, we observed a 7% decrease in FGMs. Additionally, we observed support for both an additive anthropogenic stress hypothesis and a hypothesis that stopovers act as physiological refugia, wherein individuals sampled far from stopover sites exhibited 341% higher FGM levels if in areas of low landscape integrity compared to areas of high landscape integrity. Overall, our findings indicate that the physiological state of mule deer during migration is influenced by both anthropogenic disturbances and their ability to track high-quality forage. The availability of stopovers, however, modulates physiological responses to those stressors. Thus, our results support a recent call for the prioritization of stopover locations and connectivity between those locations in conservation planning for migratory large herbivores.Rapid climate and human land-use change may limit the ability of long-distance migratory herbivores to optimally track or ‘surf’ high-quality forage during spring green-up. Understanding how anthropogenic and environmental stressors influence migratory movements is of critical importance because of their potential to cause a mismatch between the timing of animal movements and the emergence of high-quality forage. We measured stress hormones  to test hypotheses about the effects of high-quality forage tracking, human land-use and use of stopover sites on the physiological state of individuals along a migratory route. We collected and analysed FGM concentrations from 399 mule deer ( Cervus elaphus) than environmental cues . Similarly, disturbances along the migration route can negatively impact migratory movements, where animals move rapidly through or avoid areas of high human disturbance  provides a tool to evaluate how multiple potential changes in extrinsic conditions influence the internal state of migratory species. While we are not aware of any previous attempts to monitor the stress physiology of long-distance migratory herbivores along their entire migration route, groups of large herbivores with elevated glucocorticoid stress hormones have been shown to exhibit altered movement patterns at both coarse  production, we hypothesize that three primary spatially and temporally dynamic factors influence GC production for long-distance migratory herbivores Table . First, To test these hypotheses, we assessed non-invasive fecal glucocorticoid metabolite (FGM) data from a migratory mule deer population in western Wyoming, USA. This population is known to exhibit stopover behavior as well as to navigate several anthropogenic and naturally occurring bottlenecks with varying levels of human disturbance  hills and basins that lead up to the base of the Wind River Range. The approximately 40-km long section begins with the crossing of Highway 28 (two-lane highway with average daily traffic volume (ADTV) of 1 238 in 2016  and is  in 2016 ), prior  in 2016 ), to reaWe conducted field surveys and collected fresh fecal samples for stress hormone analysis during the spring (northward) migration in 2015 (14–27 April ) and 2016 (16–24 March). Given that mule deer along this migratory route exhibit a range of migratory strategies , we selected these periods during the peak of migratory activity in an effort to collect samples over a range of times and locations from deer along the entire Red Desert to Hoback (RDH) migratory route. To collect fresh samples (<24 h old), we systematically traveled portions of the migratory route each day and opportunistically observed mule deer. We never tracked the same portion of the route on consecutive days and thus avoided sampling deer we might have unintentionally disturbed. Upon encountering deer , we watched them at a distance >100 m until they moved out of view; then walked the area in ~5 m transects to locate and collect fresh fecal samples. Only samples <24 h old were collected based on seeing the defecation occur, or on the appearance and texture of the pellets. Samples were almost always clustered in discrete piles, and we attempted to only collect one sample from each individual based on a combination of pile location, sample age and/or bolus size. Samples were labeled, stored on ice in the field and within 8 h placed in a −80°C freezer prior to shipment to the laboratory.g for 20 min, and the supernatant stored at −84°C until assayed.We placed frozen fecal samples in a lyophilizer  for 24 h. Freeze-dried samples were sifted through a stainless steel mesh to remove large debris, with ~0.20 g placed in a test tube with 2.0 ml of 90% methanol and vortexed at high speed in a multi-tube vortexer for 30 min. Samples were centrifuged at ~1900 We used I125 corticosterone radioimmunoassay (RIA) kits  previously validated for use with white-tailed deer , inter-assay variation was 6.7% and average intra-assay variation was 1.4%. For fecal samples collected and analysed in 2016 (n = 170), inter-assay variation was 6.3% and average intra-assay variation was 1.0%.Assay sensitivity was 1.25 ng/g. The manufacturer’s reported cross-reactivity of the antisera was 100% with corticosterone and <1% for other steroids. For fecal samples collected in 2015 .Second, long-distance migratory herbivores also make fine-scale movement decisions along their migration route in an effort to track peak forage quality  and days to peak IRG.Glucocorticoid production is known to oscillate within a range of values in response to a suite of intrinsic  and extrinsic  stressors, yet stochastic events or the introduction of novel stressors can lead to elevated GC levels . In largLoxodonta africana) exhibit elevated GC concentrations and rapidly move through or otherwise avoid areas associated with human disturbance in favor of forested areas that act as refugia (Antilocapra americana) delay progress along their migration route just ahead of anthropogenic barriers (e.g. fences) or bottlenecks  and fixed and random effects  using the ‘MuMIn’ package in Program R , which was surprising because anthropogenic disturbance has been demonstrated to influence the stress axis of a wide variety of vertebrate taxa , includiOverall, given the rapidly increasing rate of land-use change along corridors for many long-distance migratory species across the globe , there iIn conclusion, our study of FGMs in a long-distance migratory herbivore allowed us to gain several important, novel insights into the response of individuals to human activity along their migration corridor. This initial correlative study provides support for further, direct experimental testing of the mechanistic role GC might play in animal decision making during migration. For example, a key next step is to use GPS-tracking to link individual behavior to physiology throughout a migration and across differing migration strategies and seasons. Regardless, our findings suggest that during spring migration, populations appear to be responding to forage quality and anthropogenic disturbance, both individually and interactively, and that the availability of stopovers likely reduces physiological stress. Further, our findings suggest that monitoring of FGM concentrations along a migration route can be used not only to target areas where conservation action might be needed for migratory herbivores, but also to evaluate the success of conservation actions. More generally, given the threats that animal migrations face across the globe, our work highlights the potential for future pairing of movement and remote-sensing data with non-invasive physiological monitoring to help better understand migratory behavior and to guide future conservation efforts.


Diffusion magnetic resonance imaging and tractography has an important role in the visualization of brain white matter and assessment of tissue microstructure. There is a lack of correspondence between diffusion metrics of live tissue, ex vivo tissue, and histological findings. The objective of this study is to elucidate this connection by determining the specific diffusion alterations between live and ex vivo brain tissue. This may have an important role in the incorporation of diffusion imaging in ex vivo studies as a complement to histological sectioning as well as investigations of novel neurosurgical techniques.This study presents a method of high angular resolution diffusion imaging and tractography of intact and non-fixed ex vivo piglet brains. Most studies involving ex vivo brain specimens have been formalin-fixed or excised from their original biological environment, processes both of which are known to affect diffusion parameters. Thus, non-fixed ex vivo tissue is used. A region-of-interest based analysis of diffusion tensor metrics are compared to in vivo subjects in a selection of major white matter bundles in order to assess the translatability of ex vivo diffusion measurements.Tractography was successfully achieved in both in vivo and ex vivo groups. No significant differences were found in tract connectivity, average streamline length, or apparent fiber density. Significantly decreased diffusivity  in the non-fixed ex vivo group and unaltered fractional anisotropy (p>0.059) between groups were observed.This study validates the extrapolation of non-fixed fractional anisotropy measurements to live tissue and the potential use of ex vivo tissue for methodological development. Diffusion magnetic resonance imaging (dMRI) has become widely used to non-invasively study neuroanatomical connectivity and white matter microstructure. Ex vivo specimens can be important tools for dMRI investigations as voxel resolutions on the scale of hundreds of μm can be achieved while in vivo dMRI is limited to voxel sizes in the range of 1–3 mm –6. This DMRI is based upon sensitivity to the movement of water in tissue, particularly in white matter where diffusion is anisotropic. Fiber tractography techniques can be used to study connectivity via three-dimensional reconstruction of white matter tracts. Quantitative assessment of selected tracts via diffusion tensor imaging (DTI) metrics can provide insight into white matter microstructure. Fractional anisotropy (FA) is a commonly used measure which broadly describes fiber integrity and is sensitive to local variations in myelination, crossing fiber orientation, and axonal density . Axial  placement.These experiments, including the animal handling and procurement procedures, were approved by the Animal Care Committee at the Hospital for Sick Children in Toronto, ON, Canada and follow the standards set out by the Canadian Council on Animal Care (CCAC).Twenty-one male Yorkshire piglets  were used in this study . Animals undergoing in vivo imaging  were housed in a temperature- and humidity-controlled facility with a 12-hour light/dark cycle and fed with commercial piglet milk replacer. Prior to MR scanning, piglets were pre-anesthetized with reconstituted ketamine [11.5 mg/kg IM] , intubated, and maintained under anesthesia with 2.5% isoflurane and 2 L oxygen supplied by an MR-compatible ventilator system. Piglets were positioned prone and face first in the magnet bore as schematically shown in Nine ex vivo specimens were used in this study, independent from the in vivo cohort . Researchers were not involved in the sacrifice of ex vivo animal samples. Immediately following euthanasia the piglet heads were removed and cooled in a 4°C refrigerator in order to limit tissue decomposition by autolysis or bacterial degradation . No otheImaging was performed on a 3T scanner (Philips Achieva) using a 32-channel receive-only head coil placed around the subject head, using the same protocol for both subject groups. Three-dimensional anatomical T1-weighted magnetization prepared gradient echo (MPRAGE) images were acquired with the following parameters: repetition time (TR) 8.15 ms; echo time (TE) 3.72 ms; flip angle 8°; matrix 224 x 224; field of view (FOV) 224 mm x 224 mm; slice thickness 1.00 mm; slice number 60; bandwidth 191 Hz/pixel; acquisition duration 14 min 35 s.b-value of 800 s/mm2 along 128 directions. Additional parameters were: TR 5844.97 ms; TE 105.90 ms; flip angle 90°; matrix 128 x 128; FOV 205 mm x 205 mm; slice thickness 1.60 mm; slice number 38; SENSE reduction factor 2; diffusion gradient pulse duration 15.7 ms, pulse time interval 52.9 ms; bandwidth 1431 Hz/pixel; acquisition duration 29 min 33 s. Additional baseline images with b-value = 0 s/mm2 were acquired in both forward and reverse phase encoding directions (one in each direction). These baseline images were used in the image post-processing stage to estimate the susceptibility-induced off-resonance fields.High angular resolution diffusion-weighted images (HARDI) were collected using a spin-echo single-shot echo-planar imaging (EPI) sequence with a https://fsl.fmrib.ox.ac.uk/fsl/fslwiki) [A schematic overview of the diffusion data analysis is shown in fslwiki) –31. The fslwiki) . This meFollowing these distortion corrections, diffusion tensors were calculated using an iteratively reweighted linear least squares estimator . Tensorshttps://www.florey.edu.au/imaging-software). Non-brain tissue was excluded from fiber tracking using a mask generated from the corrected DWI image [Fiber tracking was performed using MRtrix  were measured for each generated tract and compared across groups. These quantitative tract comparisons, as well as qualitative assessment of anatomical connectivity, serve as quality assurance for the image and tract quality in guiding region of interest (ROI) placement. Significant tractography differences between groups might suggest that these images are not reliable for ROI guidance and metric analysis.A ROI-based analysis was performed to extract scalar metrics  from voxels containing fiber projections. Separate ROIs were used for subregions and bilateral projections  in order to capture local variability in diffusion metrics. Tractography was used for guidance in ROI placements to provide assurance that metrics are extracted from the selected white matter bundles and minimize infiltration from adjacent grey matter, cerebrospinal fluid, or white matter of non-interest. ROIs were drawn in template space and transformed to individual subject space for metric extraction in order to limit experimenter bias and give equal weight to each subject’s contribution to the group analysis. The corpus callosum was subdivided into five vertical partitions in the anterior-posterior extent as proposed by Hofer and Frahm . The fort-tests with temperature as a covariate. Shapiro-Wilk tests of normality were used to confirm that the data is normally distributed. Levene’s test was used to verify equivalence of variance. For data where the assumption of homogeneity of variances is not satisfied, Welch’s analysis of variance is performed. A Bonferroni correction for multiple comparisons resulted in an adjusted alpha threshold of p<0.003. A power analysis was performed using a confidence level of 5% and statistical power level of 80% resulting in an independent sample size of 9.Group comparisons of DTI metrics between in vivo and ex vivo ROIs were performed using two-tailed, independent sample Quantitative assessment of diffusion metrics were performed for each ROI across both groups . SignifiConsistent differences in anisotropy were found across the five subregions of the corpus callosum in both in vivo and ex vivo groups. A trend was observed where FA values in the extreme anterior and posterior subregions  were higher than those in the middle subregions . This is similar to previous findings of  and suggVisual reconstruction of selected white matter structures was successfully performed in all imaged in vivo and ex vivo piglet brains. Qualitative inspection revealed no differences between groups with regard to anatomically accurate projections or spurious fibers. No bundle in any subject failed to meet the threshold of 1000 generated fiber counts per tract and did not exceed 1152 fiber seed attempts, well below the default maximum attempt number of 100,000. Quantitative comparison showed no significant differences between groups in average tract length or apparent fiber density .The optic fibers  could beThe trigeminal nerves  were recTracking seeds placed along the median sagittal section of the corpus callosum produced tracts which covered the entirety of the structure from the anterior portion of the genu to the posterior aspect of the splenium . ConnectTracts of the body and column of the fornix were well visualized . The bodThis study highlights the utility of DTI in ex vivo, non-fixed piglet brains. Specifically, we demonstrate that FA, considered the overall assessment of fiber integrity, is preserved for live and non-fixed ex vivo brain tissue despite a 65–88% reduction in overall water diffusion. Measures of MD, AD, and RD were significantly decreased ex vivo. Robust tractography of a variety of white matter bundles is achievable in both in vivo and ex vivo brains with no significant differences in average tract length or apparent fiber density. Reconstructed tracts were produced using identical diffusion post-processing and tracking methods. This validates the extrapolation of anisotropy measurements in ex vivo animal models to in vivo situations.Our study demonstrates that FA remained unchanged despite significant reductions to all three diffusion tensor eigenvalues post-mortem. It is inferred that the tensor eigenvalues decrease proportionally in all directions such that the directionality of diffusion is unaffected. The presumed biological components of diffusion anisotropy are the cylindrical axon microtubules and neurofilaments . These sb-values may induce differences in diffusion measurements [Previous studies have reported mixed findings on whether FA is preserved –16,19 orurements ,49. AnimAldehyde fixatives stabilize tissue metabolically and structurally by cross-linking protein amine groups throughout intra- and extracellular spaces . This teOur diffusivity measurements of MD, AD, and RD are significantly decreased from their in vivo counterparts and thus cannot be translated freely between groups. A substantial factor leading to diminished overall diffusivity is likely the decreased temperature of scanned ex vivo tissues. Le Bihan  previousAn additional process affecting our diffusivity observations could involve axonal transport, the process by which cellular organelles are moved to and from the neuron cell body via axon microtubules. Absolute diffusion rates may be aided by this process in a living organism and presumably hindered ex vivo. Axonal transport has been evaluated for its role in anisotropic diffusion in excised garfish nerves that were treated with vinblastine, a substance which depolymerizes microtubules and inhibits axonal transport . The autOur study agrees with previous observations in both fixed and non-fixed ex vivo nervous tissue where diffusivity is significantly decreased by 30% or more –21,23,25Since tissue fixation processes either halt or substantially slow down the metabolic decay process, careful consideration must be taken regarding post-mortem interval (PMI), the time from death to fixation, and SI, the time from death to image acquisition. Previous fixation studies show a heavy correlation of PMI and, to a lesser degree, SI with decreases in FA and diffusivity measures ,12. In bThese degenerative processes may be mitigated somewhat in non-human research studies where tissues may be fixed via transcardial perfusion shortly after ex vivo status, resulting in small PMI, in addition to immersion fixation following tissue extraction. Cardiac injection may be performed pre-mortem in some cases, resulting in PMI of zero . A proceWith regard to human samples, this perfusion fixation approach is generally not possible. In humans, immediate transcardial perfusion at death is not possible, resulting in long PMI and thus more time for tissue degeneration to occur. Often, immersion fixation is the only possible avenue for fixation in human specimens, resulting in poorly fixed tissue due to the longer time scales for fixative compounds to passively diffuse into the tissue from the outside in, effectively increasing the PMI for interior tissue .Because these decay processes are limited by cold temperatures, our ex vivo specimens were stored in a 4°C refrigerator before scanning. A previous study whose authors employed similar sample handling methods found no correlation between FA measurements and SI over a mean 40 hour period in non-fixed human brains . These rOur findings do not disqualify the use of fixed tissue samples for studies of tissue cytoarchitecture or white matter connectivity. Rather, we wish to highlight the potential translational value of non-fixed tissue as diffusion imaging becomes more ubiquitous in the study of neural microstructure and connectivity. Indeed, fixed tissue samples offer the advantage of temporal stability for time-dependent improvements in image resolution allowing detailed analysis of structure at the micron scale.b-value [However, diffusion-based investigations of ex vivo tissue may be confounded in their extrapolation to in vivo biological environments by the decision to study fixed or non-fixed tissue and the manner in which fixation was performed. The findings reported in this study indicate that anisotropy measurements and tractography may be translated from non-fixed ex vivo to live tissue. This validates the use of ex vivo animal models for methodological or therapeutic development studies which incorporate diffusion imaging without the need for tissue fixation. FA is often cited as a defining metric in comparisons between pre- and post-surgical assessment and between disease and control groups. A successful surgical treatment may be defined in part by its ability to restore altered FA measurements in a patient to that of a healthy non-patient group. Novel neurosurgical procedures, for example, require thorough characterization before use in living humans. Phantoms or fixed tissue specimens may provide stable imaging but may not be a good model for the structural complexity or physical characteristics of live tissue. In these cases the translatability of such pre-clinical development and image characterization is hindered. Live animal studies are certainly a viable alternative but these models carry significant logistical and financial burden. Ex vivo animal specimens may circumvent these hurdles in lieu of live subjects. The processing methods presented here are identical between groups. However, scanning protocols may need to be modified for ex vivo subjects in order to account for decreased diffusivity measurements, namely by increasing the diffusion gradient b-value .Unaltered FA was found between both groups which had otherwise not been treated or altered by exogenous forces. We therefore cannot comment on the ability of FA changes resulting from externally applied procedures in one group to translate identically to the other after the same treatment. With regard to tractography, the tract subregions of interest were defined manually and thus comparisons of average tract length and apparent fiber density between live and ex vivo groups apply only to fully generated tracts. Local tractography variations may exist but are averaged over the entire tract. Subregions of the corpus callosum were defined according to cortical projections observed in humans. These subdivisions may not be appropriate for the cortical connectivity in porcine brains. We refer readers to work by Knosche et al. on downstream cortical tracing in pig brains .Acquisition parameters were identical between groups in the present work, which is a limitation due to the significantly decreased diffusivity observed and its related detrimental effects on SNR. It is suggested that researchers select an appropriately increased b-value for ex vivo scanning (relative to in vivo) to account for decreased diffusivity and signal intensity observed. We refer readers to a recent review article by Roebroeck et al. for a discussion on appropriate scanning and tissue preparation protocols in ex vivo imaging .The ex vivo status of tissue results in important changes in the diffusion MRI properties and microstructure of the tissue. These changes can be further distinguished between non-fixed tissue and aldehyde-fixed tissue where some diffusion measurements may be preserved in the former but may not be in the latter. Researchers may thus find value in the use of non-fixed specimens to optimize procedures and treatment parameters with an understanding of the translatability of diffusion anisotropy observations to in vivo subjects.


The upper incisors torque expression is essential for the orthodontic treatment accuracy. Various orthodontic devices are claimed to have different inclination control capacity. The purpose of this retrospective study was to compare the radiographic buccolingual inclination of upper incisors in patients treated with three different orthodontic techniques.∧SnaSnp, 11∧Ocl and I+ TVL. All variables were measured before (T0) and after (T1) treatment and their variation over treatment was assessed. Conventional brackets , self-ligating appliances , and aligners  were tested. Cephalometric data of 25 patients with similar skeletal and dental pretreatment parameters were collected for each technique. Position changes of upper central incisors were assessed with radiographic evaluation before and after therapy. Three different parameters were considered: 11∧SnaSnp and 11∧Ocl angles showed the highest numeric variation with conventional brackets. Lowest values were reported with aligners. However, the differences among various techniques were not significant for both angles (P>0.05). Also I+ TVL linear value variation did not show significant differences among the different groups tested (P>0.05). When evaluating angular measurements, 11 Conventional multibrackets appliance showed the highest incisal position variations over treatment, but the differences among various groups were not significantly different. The expression of torque is one of the most important key factors in orthodontic treatment. Clinically, the torque represents the third key of occlusion, described as the buccolingual inclination of the dental crown . The optThe conventional multibrackets technique is the most widely studied orthodontic appliance and it has been described as a tool with efficient torque control capability , 5. SincIn order to reduce chairside time and bracket/wire friction, self-ligating brackets have been introduced in orthodontic practice –10. ThesThe growing aesthetic demands of orthodontic patients led the research towards the development of more aesthetics and comfortable orthodontic appliances . In the Despite claims about the capability of aligners, some authors state that evidence is generally lacking. Shortened treatment duration and chair time in mild-to-moderate cases appear to be the only significant effectiveness of aligners . Even ifTherefore, the aim of this retrospective clinical study was to evaluate the effectiveness of conventional brackets, self-ligating bracket, and aligners on torque expression, evaluating lateral cephalometric values in an orthodontic population. The null hypothesis of the study was the absence of significant difference in incisal inclination variation during therapy among the three different techniques.Three different orthodontic techniques were evaluated: conventional brackets , self-ligating appliances , and aligners .The present study followed Helsinki Declaration. Internal Unit Review Board accepted the study design (Ref: 16-0318).25 patients for each technique were retrospectively selected .Inclusion criteria involved permanent teeth, dental class I, or mild classes II and III and need for upper incisal torque change. Exclusion criteria were the presence of anterior or posterior cross bites, skeletal class III, severe skeletal deformity, previous orthodontic therapies, skull/facial traumas, and temporomandibular disorders.Maximum torque expression was obtained by a 0.019x0.025-inch stainless steel archwires  for conventional and self-ligating brackets, whereas for aligners by the Power Ridge technology .For each patient, lateral cephalometric radiographs before (T0) and after therapy (T1) were collected and computerized cephalometric traces were performed by the same operator twice, with an interval of two weeks .An expert operator in the technique treated every group of patients.The following points were considered for each patient's radiographs: upper incisal point (INI+), upper apical point (API+), anterior nasal spine (Sna), posterior nasal spine (Snp), occlusal anterior point (Ocla), occlusal posterior point (Oclp), gonion (Go), menton (Me), orbitale (Or), porion (Po), sella (S), A point (A), and B point (B). For each X-ray, the following plans were traced: upper incisal axis (INI + - API +); palatal plane (Sna - Snp); occlusal plan (Ocla - Oclp); mandible plane (Go - Me); Frankfurt horizontal plane (Or - Po); TVL ; S-N; N-A; and N-B planes. The same operator performed all the tracings.∧GoMe (to identify the divergence).The following angles were employed in order to evaluate the homogeneity of the patients enrolled and to eliminate possible sample bias: ANB and Wits index ; SnaSnp∧Snp ; 11∧Ocl ; and I + TVL (linear distance of the most advanced point of the vestibular surface of the upper incisor from the TVL).The torque evaluation was performed testing the variation over treatment of the following variables: 11-SnaData analysis was performed with software . To estimate the method error, the same operator retraced radiographs after a period of 6 weeks, measures were assessed with t-test, and no significant variations were reported between two groups. Descriptive statistic of the different variables  was computed for each group. The normality of distributions was evaluated by Kolmogorov and Smirnov tests. Subsequently an ANOVA test was applied followed by the Tukey test as a post hoc test. For all tests the significance was set for P <0.05.No significant differences (P>0.05) were observed among the three different groups when evaluating pretreatment skeletal and dental parameters .The variation of the upper incisal inclination and, consequently, the expression capacity of the upper incisors torque was evaluated at T0 and T1 using three different values:  + - TVL . The var∧SnaSnp and 11∧Ocl angles showed the highest numeric variation with conventional brackets. Lowest values were reported with aligners (When evaluating angular measurements, 11aligners . Howeveraligners  did not The main objective of the study was to evaluate the ability to control the buccolingual inclination of upper incisors with three different orthodontic appliances: conventional brackets, self-ligating braces, and aligners. The null hypothesis of the present report was accepted.∧SnaSnp and 11∧Ocl angles have been evaluated. These angles measure the inclination of the upper incisors, respectively, on the bispinal plane and on the occlusal plane. The values measuring their modification over treatment were compared. For both angles, conventional brackets showed the highest numeric variation . Lower values were reported with self-ligating devices . The lowest data were reported with aligners . However, the differences among various techniques were not significant for both angles. Moreover, also the variation during treatment of the linear measurement I+ TVL showed no significant difference among the three different techniques. In the present study, all the data were measured with cephalometric software that allows an increased precision in landmarks localization and provides decimal rounded measurements, easing an accurate evaluation and comparison of clinical characteristics  and linear [I+ TVL] modifications of the radiographic position of the upper incisor occurring during treatment are important indicators to measure the clinical reliability and efficiency of orthodontic appliances in controlling upper incisors. Poor variations of these indicators may result from information loss due to devices inaccuracy [The angular [11The first orthodontic technique studied in the present report was the conventional multibrackets appliance. In this system, torque control depends on many clinical factors such as initial tooth inclination, vertical bracket position, and tooth anatomy . Orthodo∧SNASNP and 11∧Ocl variations were lower than conventional brackets, even if the differences were not statistically significant.The second orthodontic devices evaluated in the present report were self-ligating brackets. These stainless steel brackets are characterized by the presence of a fourth moving wall that converts the slot into a tube . Self-liThe third technology studied in this research was aligners. This technique has some considerable clinical advantages. In fact, these devices allow satisfying the patient's aesthetic demand, due to their small size and transparency . MoreoveThe measurements of the buccolingual inclination in the digital world allowed previous authors to state that, when a mean root torque information of 10.4° was required for an upper central incisor, an expression of the 99% of the third-order information was detected, demonstrating a negligible torque loss . DespiteOther authors analyzed cephalometric position of mandibular incisors . In mildWhen evaluating torque expression of orthodontic appliances also the age of the patients has to be carefully considered. In fact it is well known that torque and overjet change remarkably during certain growth phases , 36. In Finally, the existing literature is mainly focused on materials and methodological aspects of aligner orthodontics, while only in the last years the interest in evaluating the efficiency of orthodontic movements obtained by these devices has grown very fast. However, the number of studies comparing the effects of different orthodontic techniques, including the aligners, is very low. Other studies have evaluated torque control efficiency of different orthodontic technique, analyzing the position of teeth in vitro , 10, 25.This study showed that both aligners and self-ligating brackets generated lower torque control if compared to conventional brackets, but these differences were not statistically significant different. Further studies are needed to confirm or disprove the results of this retrospective research.∧SnaSnp and 11∧Ocl angles showed the highest numeric variation with conventional brackets. Lowest values were reported with aligners. However, the differences among various techniques were not statistically significant for both angles. Also I+ TVL linear value variation did not show significant differences among the different groups tested.When evaluating radiographic angular measurements during treatment, 11Based on these results, all the three different systems showed good clinical reliability in the upper incisor torque control.