Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.
25 Related JoVE Articles!
Gene Transfer for Ischemic Heart Failure in a Preclinical Model
Institutions: Mount Sinai School of Medicine .
Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.
Medicine, Issue 51, Myocardial infarction, Gene therapy, Intracoronary injection, Viral vector, Ischemic heart failure
Growth of Mycobacterium tuberculosis Biofilms
Institutions: University of Pittsburgh, University of Pittsburgh.
, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis
is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1
, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4
. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6
. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7
In a series of recent papers we established that M. tuberculosis
and Mycobacterium smegmatis
have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10
. M. tuberculosis,
however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9
. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis
is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis
biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis
, with deletion in the two loci, panCD
that are critical for in vivo
growth of the pathogen 9
. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.
Immunology, Issue 60, Mycobacterium tuberculosis, tuberculosis, drug tolerance, biofilms
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia
Institutions: New York Institute of Technology College of Osteopathic Medicine.
Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071
and resulted in the hospitalization of 1.1 million patients2
. With an average length of in-patient hospital stay of five days2
, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053
. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4
. (Figure 1)
Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients’ length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5
. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6
. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10
The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11
Medicine, Issue 87, Pneumonia, osteopathic manipulative medicine (OMM) and techniques (OMT), lymphatic, rib raising, thoracic pump, muscle energy, doming diaphragm, alternative treatment
Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
Institutions: National Cancer Institute.
Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.
Immunology, Issue 86, HIV, cytotoxicity, infectivity, luciferase, drug resistance, integrase, reverse transcriptase
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Institutions: NCI-Frederick, University of Pittsburgh, Tuffts University.
Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART).
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19
and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15
,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19
. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.
Immunology, Issue 55, single genome sequencing, SGS, real-time PCR, single-copy assay, SCA, HIV-1, ultra-sensitive, RNA extraction
Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients
Institutions: Hadassah Hebrew-University Medical Center.
In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.
Medicine, Issue 86, Optic neuritis, visual impairment, dynamic visual functions, motion perception, stereopsis, demyelination, remyelination
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro
, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans
. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo
, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
Institutions: Cedars-Sinai Medical Center.
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
Neuroscience, Issue 88, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
Bronchial Thermoplasty: A Novel Therapeutic Approach to Severe Asthma
Institutions: Virginia Hospital Center, Virginia Hospital Center.
Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.
Medicine, Issue 45, bronchial thermoplasty, severe asthma, airway smooth muscle, bronchoscopy, radiofrequency energy, patient management, moderate sedation
Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage
Institutions: Duke University, Duke University, Duke University, Duke University.
Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain's response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.
Medicine, Issue 89, intracerebral hemorrhage, mouse, preclinical, autologous blood, collagenase, neuroscience, stroke, brain injury, basal ganglia
The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis
Institutions: University of Southern California, University of California, San Francisco , University of California Irvine School of Medicine, University of Arizona College of Medicine, Chicago College of Osteopathic Medicine.
Psoriasis is a chronic, immune-mediated inflammatory skin disease affecting approximately 2-3% of the population. The Goeckerman regimen consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). Goeckerman therapy is extremely effective and relatively safe for the treatment of psoriasis and for improving a patient's quality of life. In the following article, we present our protocol for the Goeckerman therapy that is utilized specifically at the University of California, San Francisco. This protocol details the preparation of supplies, administration of phototherapy and application of topical tar. This protocol also describes how to assess the patient daily, monitor for adverse effects (including pruritus and burning), and adjust the treatment based on the patient's response. Though it is one of the oldest therapies available for psoriasis, there is an absence of any published videos demonstrating the process in detail. The video is beneficial for healthcare providers who want to administer the therapy, for trainees who want to learn more about the process, and for prospective patients who want to undergo treatment for their cutaneous disease.
Medicine, Issue 77, Infection, Biomedical Engineering, Anatomy, Physiology, Immunology, Dermatology, Skin, Dermis, Epidermis, Skin Diseases, Skin Diseases, Eczematous, Goeckerman, Crude Coal Tar, phototherapy, psoriasis, Eczema, Goeckerman regimen, clinical techniques
Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Institutions: University of Arizona, University of Arizona.
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2
. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
Medicine, Issue 77, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Institutions: Louisiana State University Health Sciences Center.
There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice
Institutions: San Raffaele Scientific Institute, Italian Cystic Fibrosis Research Foundation.
A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa
agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo
. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa
and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa
clinical strains in the agar-beads in vitro
, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa
RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa
colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.
Infection, Issue 85, Opportunistic Infections, Respiratory Tract Infections, Inflammation, Lung Diseases, Cystic Fibrosis, Pseudomonas aeruginosa
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model
Institutions: University Medical Center Utrecht, Vascular Connect b.v., University Medical Center Utrecht, University Medical Center Utrecht.
To simplify and facilitate beating heart (i.e.,
off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e.,
occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.
Medicine, Issue 93, Anastomosis, coronary, anastomotic connector, anastomotic coupler, excimer laser-assisted nonocclusive anastomosis (ELANA), coronary artery bypass graft (CABG), off-pump coronary artery bypass (OPCAB), beating heart surgery, excimer laser, porcine model, experimental, medical device
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale
Institutions: Brown University, Women & Infants Hospital of Rhode Island, University of Massachusetts, Boston.
There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.
Behavior, Issue 90, NICU Network Neurobehavioral Scale, NNNS, High risk infant, Assessment, Evaluation, Prediction, Long term outcome
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Modified Technique for Coronary Artery Ligation in Mice
Institutions: Sahlgrenska Academy, University of Gothenburg.
Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3
. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4
. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5
. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.
Medicine, Issue 73, Anatomy, Physiology, Biomedical Engineering, Surgery, Cardiology, Hematology, myocardial infarction, coronary artery, ligation, ischemia, ECG, electrocardiology, mice, animal model
Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging
Institutions: Brigham and Woman's Hospital, Brigham and Woman's Hospital.
Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo
fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105
cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo
fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.
Developmental Biology, Issue 31, Mouse, skeletal muscle, in vivo, fluorescence imaging, cell therapy, longitudinal monitoring, quantification
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research
Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment
Institutions: Havard Medical School, Massachusetts General Hospital.
Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel. Surgical clipping is an important treatment for aneurysms which attempts to exclude blood from flowing into the aneurysmal segment of the vessel while preserving blood flow in a normal fashion. Improper clip placement may result in residual aneurysm with the potential for subsequent aneurysm rupture or partial or full occlusion of distal arteries resulting in cerebral infarction. Here we describe the use of an ultrasonic flow probe to provide quantitative evaluation of arterial flow before and after microsurgical clip placement at the base of a middle cerebral artery aneurysm. This information helps ensure adequate aneurysm reconstruction with preservation of normal distal blood flow.
Medicine, Issue 31, Aneurysm, intraoperative, brain, surgery, surgical clipping, blood flow, aneurysmal segment, ultrasonic flow probe