Large-scale characterization of the microvascular geometry in development and disease by tissue clearing and quantitative ultramicroscopy

Three-dimensional assessment of optically cleared, entire organs and organisms has recently become possible by tissue clearing and selective plane illumination microscopy (“ultramicroscopy”). Resulting datasets can be highly complex, encompass over a thousand images with millions of objects and data of several gigabytes per acquisition. This constitutes a major challenge for quantitative analysis. We have developed post-processing tools to quantify millions of microvessels and their distribution in three-dimensional datasets from ultramicroscopy and demonstrate the capabilities of our pipeline within entire mouse brains and embryos. Using our developed acquisition, segmentation, and analysis platform, we quantify physiological vascular networks in development and the healthy brain. We compare various geometric vessel parameters (e.g. vessel density, radius, tortuosity) in the embryonic spinal cord and brain as well as in different brain regions (basal ganglia, corpus callosum, cortex). White matter tract structures (corpus callosum, spinal cord) showed lower microvascular branch densities and longer vessel branch length compared to grey matter (cortex, basal ganglia). Furthermore, we assess tumor neoangiogenesis in a mouse glioma model to compare tumor core and tumor border. The developed methodology allows rapid quantification of three-dimensional datasets by semi-automated segmentation of fluorescently labeled objects with conventional computer hardware. Our approach can aid preclinical investigations and paves the way towards “quantitative ultramicroscopy”.     

Diuretic-induced hyponatremia and osteoporotic fractures in patients admitted to the emergency department

Objective

Hyponatremia is a complication of diuretic treatment and has been recently identified as a novel factor associated with osteoporosis and fractures. The impact of diuretic-associated electrolyte disorders on osteoporotic fractures (OF) has rarely been studied systematically.

Design and setting

We conducted a study in patients presenting to the emergency department at the University Hospital Bern. In this retrospective case series analysis of prospectively gathered data, over a 2-year period we identified 10,823 adult (≥50 years) outpatients with a measured baseline serum sodium, at admission to the hospital. OF patients were compared to a control group without fractures using standard statistical methods.

Results

Four hundred and eighty (5%) patients had 547 OF. The OF group had a higher mean age (73 vs. 68 years, p < 0.0001), smaller proportion of men (37% vs. 58%, p < 0.0001), higher hospitalisation rate (83% vs. 62%, p < 0.0001) and longer hospital stay (8 vs. 6 days, p < 0.0001). Any diuretic agent (p < 0.0001), loop diurietics (p = 0.02), spironolactone (p = 0.02) and amiloride (p < 0.01) were used significantly more in OF patients, but not thiazides (p = 0.68). The prevalence of hyponatremia increased significantly (p < 0.0001) with the number of diuretics taken. Advanced age (odds ratio [OR] 1.04, p < 0.0001), hyponatremia (OR 1.46, p = 0.011) higher serum creatinine (OR 1.53, p = 0.0001), furosemide use alone (OR 1.40, p = 0.01) and co-treatment with amiloride (OR 2.22, p = 0.02) were associated with a higher risk for OF.

Conclusions

This study highlights the clinical association of hyponatremia during the use of certain diuretics (i.e. furosemide or in combination, i.e. amiloride) with an increased risk of osteoporosis associated fractures. Although evidence-based data is currently lacking a pragmatic approach concerning hyponatremia monitoring and correction appears reasonable in selected groups of patients.     

Emerging therapies for postmenopausal vaginal atrophy

Symptoms related to vaginal atrophy are a significant problem for postmenopausal women and estrogen has been the gold standard for its treatment. A number of recent reviews of vaginal estrogen products are available. This review will, therefore, focus on other products and potential products for this indication, including the tissue selective estrogen complex and selective estrogen receptor modulators. Additionally, lesser-studied approaches will be discussed.     

Translational cell biology of highly malignant osteosarcoma

Highly malignant osteosarcoma (HMO) is the most frequent malignant bone tumor preferentially occurring in adolescents and children with a second more flat peak in patients over the age of 60. The younger patients benefit from combined neoadjuvant chemotherapy with 65–70% 5-year survival rate. In patients with metastatic HMO the 5-year survival rate is consistently poor with approximately 30%. In the last several years strategies for target therapies have been developed by using next generation sequencing (NGS) for defining targetable molecular factors. However, it has so far been challenging to establish an effective target therapy for so-called ‘orphan tumors’ without recognizable driver mutations, including HMO. The molecular genetic studies using NGS have shown that HMOs are genomically unstable tumors with highly complex chaotic karyotypes. Before the background of this genetic complexity more investigations should be performed in the future for defining targetable biological factors. As the prognosis could not be improved for 40 years one may expect improvements for patients only by gaining a deeper understanding of the cell and molecular biology of HMO. The cell of origin of HMO is being clarified now. The majority of studies indicate that an osteoblastic progenitor cell is probably the cell of origin of HMO and not an undifferentiated mesenchymal stem cell. This means that the established histopathological definition of HMO through verification of osteoid production by the osteoblastic cells is well justified and will probably be the cornerstone for a precise differential diagnosis of HMO also in the years to come.     

Selective Retreatment and Sinus Lift: An Alternative Approach to Surgically Manage the Palatal Roots of Maxillary Molars

Endodontic microsurgery on the palatal root of maxillary molars presents a clinical challenge because of the root position and approximation from the maxillary sinus floor. Attempting a buccal or a palatal approach to address the root is associated with limited accessibility and visibility as well as the risk of injury to the maxillary sinus membrane and/or the greater palatine nerves and vessels. If all the maxillary molar roots require surgical intervention, two flaps may even be needed, which can make the procedure technically more difficult and lengthier. This case report presents 2 clinical cases in which apicoectomy was needed on the palatal roots of maxillary molars. The treatment includes selective nonsurgical retreatment of the palatal root and obturation using a root repair material followed by a surgical intervention from a buccal approach to treat the buccal roots, sinus lift using piezosurgery, and root resection of the palatal root. The approach was successful in both cases without any untoward events. We monitored the radiographic changes using cone-beam computed tomographic imaging immediately after the surgery and at multiple follow-up appointments. The cone-beam computed tomographic images revealed healing of the periapical disease around all the roots up to 14 and 24 months and apical repositioning of the maxillary sinus floor.     

Masthead,Volume 73, Issue 1

This review provides a summary statement of recommended implementations of arterial spin labeling (ASL) for clinical applications. It is a consensus of the ISMRM Perfusion Study Group and the European ASL in Dementia consortium, both of whom met to reach this consensus in October 2012 in Amsterdam. Although ASL continues to undergo rapid technical development, we believe that current ASL methods are robust and ready to provide useful clinical information, and that a consensus statement on recommended implementations will help the clinical community to adopt a standardized approach. In this review, we describe the major considerations and trade‐offs in implementing an ASL protocol and provide specific recommendations for a standard approach. Our conclusion is that as an optimal default implementation, we recommend pseudo‐continuous labeling, background suppression, a segmented three‐dimensional readout without vascular crushing gradients, and calculation and presentation of both label/control difference images and cerebral blood flow in absolute units using a simplified model. Magn Reson Med 73:102–116, 2015. © 2014 Wiley Periodicals, Inc.     

Partial tears of anterior cruciate ligament: Results of single bundle augmentation

Background:

Partial tears of the anterior cruciate ligament (ACL) are common and usually present with symptomatic instability. The remnant fibers are usually removed and a traditional ACL reconstruction is done. But with increased understanding of ACL double bundle anatomy, the remnant tissue preservation along with a single bundle augmentation of the torn bundle is also suggested. The purpose of this study was to evaluate the results of selective anatomic augmentation of symptomatic partial ACL tears. Our hypothesis is that this selective augmentation of partial ACL tears could restore knee stability and function.

Materials and Methods:

Consecutive cases of 314 ACL reconstructions, 40 patients had intact ACL fibers in the location corresponding to the anteromedial (AM) or posterolateral (PL) bundle and were diagnosed as partial ACL tears perioperatively. All patients underwent selective augmentation of the torn bundle, while keeping the remaining fibers intact using autogenous hamstring graft. A total of 38 patients (28 males, 10 females) were available with a minimum of 3 years followup. 26 cases had AM bundle tears and 12 cases had PL bundle tears respectively. Patients were assessed with International Knee Documentation Committee (IKDC) 2000 Knee Evaluation Form, Lysholm score; instrumented knee testing was performed with the arthrometer (KT 2000). Statistical analysis was performed to compare the preoperative and postoperative objective evaluation.

Results:

At 3 years followup, 31.6% patients were graded A, 65.8% were graded B and 2.6% was graded C at IKDC objective evaluation. Manual laxity tests, Lysholm''s score, mean side to side instrumental laxity and Tegner activity score improved significantly. 76% patients returned to preinjury level of sports activity after augmentation.

Conclusion:

The results of anatomic single bundle augmentation in partial ACL tears are encouraging with excellent improvement in functional scores, side to side laxity and return to sports activity.     

Preserving fertility in the hypogonadal patient: an update

An increasing number of young and middle-aged men are seeking treatment for symptoms related to deficient levels of androgens (hypogonadism) including depression, loss of libido, erectile dysfunction, and fatigue. The increase in prevalence of testosterone supplementation in general and anabolic steroid-induced hypogonadism specifically among younger athletes is creating a population of young men who are uniquely impacted by the testicular end-organ negative consequences of exogenous steroid use. Exogenous testosterone therapy can alter the natural regulation of the hypothalamic-pituitary-gonadal axis leading to impaired spermatogenesis with azoospermia being a serious possible result, thus rendering the individual infertile. For men of reproductive age who suffer from hypogonadal symptoms, preservation of fertility is an important aspect of their treatment paradigm. Treatment with human chorionic gonadotropin (hCG) has shown the ability not only to reverse azoospermia brought on by testosterone supplementation therapy but also to help maintain elevated intratesticular testosterone levels. In addition, selective estrogen receptor modulators, often used with hCG have been shown both to elevate total testosterone levels and to maintain spermatogenesis in hypogonadal men.     

Comparative Analysis of Immune Responses in Pigs to High and Low Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses Isolated in China

The CH‐1a and HuN4 strains of porcine reproductive and respiratory syndrome virus (PRRSV) show different pathogenicities in pigs. To understand host immune responses against these viruses, we investigated the dynamic changes in cytokine levels produced in peripheral blood of piglets infected with the highly pathogenic PRRSV HuN4 strain or the CH‐1a strain. Clinical signs, virus loads and serum cytokine levels [interferon(IFN)‐α, Interleukin (IL)‐1, TNF‐α, IL‐6, IL‐12, IFN‐γ, IL‐10 and TGF‐β] were tested. The results showed that while piglets developed effective cellular immune responses against CH‐1a infection, those infected with HuN4 displayed ineffective cellular immunity, organ lesions and persistent elevated levels of immunoregulatory cytokines (IL‐10 and TGF‐β), which delayed the development of PRRSV‐specific immune responses. These results demonstrated that HuN4 infection induced higher cytokine levels than that of CH‐1a infection induced. The changes in inflammatory cytokines intensified the inflammatory reaction and damaged the tissues and organs.     

Stimulus features coded by single neurons of a macaque body category selective patch

Body category-selective regions of the primate temporal cortex respond to images of bodies, but it is unclear which fragments of such images drive single neurons’ responses in these regions. Here we applied the Bubbles technique to the responses of single macaque middle superior temporal sulcus (midSTS) body patch neurons to reveal the image fragments the neurons respond to. We found that local image fragments such as extremities (limbs), curved boundaries, and parts of the torso drove the large majority of neurons. Bubbles revealed the whole body in only a few neurons. Neurons coded the features in a manner that was tolerant to translation and scale changes. Most image fragments were excitatory but for a few neurons both inhibitory and excitatory fragments (opponent coding) were present in the same image. The fragments we reveal here in the body patch with Bubbles differ from those suggested in previous studies of face-selective neurons in face patches. Together, our data indicate that the majority of body patch neurons respond to local image fragments that occur frequently, but not exclusively, in bodies, with a coding that is tolerant to translation and scale. Overall, the data suggest that the body category selectivity of the midSTS body patch depends more on the feature statistics of bodies (e.g., extensions occur more frequently in bodies) than on semantics (bodies as an abstract category).The body category-selective regions in the human occipito-temporal cortex are defined as those that respond to images of bodies (1–8). We previously identified two bilateral regions in the macaque inferotemporal cortex that respond stronger to monkey, human, and animal bodies in comparison with other stimuli, including faces (6). Subsequent single-unit recordings in the posterior body patch [i.e., the middle superior temporal sulcus (midSTS) body patch] demonstrated that indeed the average spiking activity of the neuron population was greater to images of bodies compared with other objects. However, the responses of single neurons showed a strong selectivity for particular body—and sometimes nonbody—images (7). However, it is still unknown what particular stimulus features single body patch neurons respond to. Moreover, we still do not know how those neurons code information about different animate and inanimate stimuli.The Bubbles technique (9), in which parts of the image of an object are sampled by trial-unique randomly positioned Gaussian apertures, has been used successfully in many psychophysical studies to reveal the features critical for certain perceptual tasks such as face identification, gender discrimination, emotional discrimination, and so forth (e.g., refs. 9–13). Although this technique has been used in neuroimaging [functional MRI (fMRI), EEG, magnetoencephalography (MEG), and electrocorticography] studies (11, 12, 14), it has rarely been exploited in single-unit studies (15, 16), and this only for face stimuli.Here, we used the Bubbles technique to reveal the image fragments that drive single midSTS body patch neurons. Bubbles provides an unbiased method for sampling the images with the advantage that it requires no prior specification of stimulus features to which the neurons are supposed to be selective. With fMRI, we first defined the midSTS body patch in two monkeys. Then, in this identified body patch we recorded the spiking activity of well-isolated single neurons in response to 100 images of various categories. Based on the spiking activity to the 100 images, we selected for each neuron a response-eliciting image. Then, we sampled the selected image at five different spatial scales with randomly positioned Gaussian apertures and recorded the responses of the neuron to a large number of these trial-unique Bubbles stimuli. Following the experiment, we applied reverse correlation to relate the excitatory and inhibitory neural responses to particular image fragments.Furthermore, we assessed whether the revealed image fragments tolerated changes in spatial location and size of the Bubbles stimuli or instead reflected spatially localized image regions. We showed before that many midSTS body patch neurons respond to silhouettes of bodies (17). Silhouettes isolate shape contours, removing texture and shading information. Thus, in a subset of neurons, we applied Bubbles to a silhouette version of the selected image.