Performing <em>In Vivo</em> and <em>Ex Vivo</em> Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...

Harvard Medical School - Beth Israel Deaconess Medical Center

Normal angiogenesis is a complex process involving the organization of proliferating and migrating endothelial cells (ECs) into a well-ordered and highly functional vascular network. In contrast, pathological angiogenesis, which is a conspicuous feature of tumor growth, ischemic diseases, and chronic inflammation, is characterized by vessels with aberrant angioarchitecture and compromised barrier function. Herein we review the subject of pathological angiogenesis, particularly that driven by vascular endothelial growth factor (VEGF-A), from a new perspective. We propose that the serious structural and functional anomalies associated with VEGF-A-elicited neovessels, reflect, at least in part, imbalances in the internal molecular cues that govern the ordered assembly of ECs into three dimensional vascular networks and preserve vessel barrier function. Adopting such a viewpoint widens the focus from solely on specific pro-angiogenic stimuli such as VEGF-A to include a key set of cytoskeletal regulatory molecules, the Rho GTPases, which are known to direct multiple aspects of vascular morphogenesis including EC motility, alignment, multi-cellular organization, as well as intercellular junction integrity. We offer this perspective to draw attention to the importance of endothelial cytoskeletal dynamics for proper neovascularization and to suggest new therapeutic strategies with the potential to improve the pathological vascular phenotype.

VEGF-A, cytoskeletal dynamics, and the pathological vascular phenotype.

Tumors, wounds, and chronic inflammatory disorders generate a new vascular supply by a process known as pathological angiogenesis. Whereas formation of the normal blood vasculature requires the interaction of many different agonists and inhibitors, including vascular endothelial growth factor-A (VEGF-A) and other members of the vascular permeability factor/VEGF family, pathological angiogenesis is a cruder, simpler process that can be replicated by a single VEGF-A isoform, VEGF-A(164/5). VEGF-A(164/5) induces the formation of several distinctly different types of new blood vessels that differ from normal blood vessels with respect to organization, structure, and function. Elucidating the properties of these new vessels has led to a better understanding of angiogenesis and will hopefully lead to new approaches to antiangiogenic therapy.

VEGF-A and the induction of pathological angiogenesis.

The vascular system has the critical function of supplying tissues with nutrients and clearing waste products. To accomplish these goals, the vasculature must be sufficiently permeable to allow the free, bidirectional passage of small molecules and gases and, to a lesser extent, of plasma proteins. Physiologists and many vascular biologists differ as to the definition of vascular permeability and the proper methodology for its measurement. We review these conflicting views, finding that both provide useful but complementary information. Vascular permeability by any measure is dramatically increased in acute and chronic inflammation, cancer, and wound healing. This hyperpermeability is mediated by acute or chronic exposure to vascular permeabilizing agents, particularly vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A). We demonstrate that three distinctly different types of vascular permeability can be distinguished, based on the different types of microvessels involved, the composition of the extravasate, and the anatomic pathways by which molecules of different size cross-vascular endothelium. These are the basal vascular permeability (BVP) of normal tissues, the acute vascular hyperpermeability (AVH) that occurs in response to a single, brief exposure to VEGF-A or other vascular permeabilizing agents, and the chronic vascular hyperpermeability (CVH) that characterizes pathological angiogenesis. Finally, we list the numerous (at least 25) gene products that different authors have found to affect vascular permeability in variously engineered mice and classify them with respect to their participation, as far as possible, in BVP, AVH and CVH. Further work will be required to elucidate the signaling pathways by which each of these molecules, and others likely to be discovered, mediate the different types of vascular permeability.

Vascular permeability, vascular hyperpermeability and angiogenesis.

Musculoskeletal deconditioning is a well-known consequence of microgravity. However, the effects of partial gravity, such as that experienced on the moon (0.16 g) or Mars (0.38 g), on musculoskeletal health remain relatively unexplored. Because Mars is being increasingly viewed as the likely next extraterrestrial site for human exploration, there is an increasing need for Earth-based models that can replicate the long-term physiological effects of microgravity. These models would also offer the opportunity to explore the potential impact of partial artificial gravity (as would be achieved by centrifugation). In this study, we describe a novel partial gravity model that can be employed in rats over extended periods of time. We demonstrate that 2 wk of partial weight bearing at 20, 40, or 70% of normal loading affects the musculoskeletal health of the animals, as evidenced by decreased trabecular bone density (ranging from -7.5 ± 2.7% at 70% of normal loading to -27.9 ± 2.9% at 20%), hindlimb muscle mass, and impaired muscle function as characterized by grip force. This new model will facilitate studies of the physiological changes occurring in partial gravity and allow for the design of potential countermeasures to mitigate these changes. NEW & NOTEWORTHY This research article describes the first quadrupedal unloading model in rats that is sustainable for investigating the physiological alterations occurring in partial gravity environments, providing a new and adaptable model for ground-based research for future space exploration.

A novel partial gravity ground-based analog for rats via quadrupedal unloading.

Many studies have investigated the physiological response to microgravity in both astronauts and animals. However, while space agencies have sought to deploy more women on their missions; animal models rarely include females studies or comparisons between males and females. Therefore, we exposed adult female rats to 2 weeks of partial weight-bearing at either 100% of their normal loading (PWB100) or 40% of their normal loading (PWB40), corresponding to Martian gravity-analog, and assess muscle function, force and histomorphometry. Females exposed to PWB showed an 11.62% decline in hindlimb grip force associated with an 11.84% decrease in soleus myofiber size after 14 days of exposure, while maintaining normal blood oxygenation and stress levels. This pilot study represents the first experiment designed to understand the muscular disuse associated with a partial reduction in mechanical loading in female rats, and the first step needed to develop successful mitigating strategies.

Partial Weight-Bearing in Female Rats: Proof of Concept in a Martian-Gravity Analog.

[This corrects the article DOI: 10.3389/fphys.2020.00302.].

Corrigendum: Partial Weight-Bearing in Female Rats: Proof of Concept in a Martian-Gravity Analog.

As astronauts prepare to undertake new extra-terrestrial missions, innovative diagnostic tools are needed to better assess muscle deconditioning during periods of weightlessness and partial gravity. Electrical impedance myography (EIM) has been used to detect muscle deconditioning in rodents exposed to microgravity during spaceflight or using the standard ground-based model of hindlimb unloading via tail suspension (HU). Here, we used EIM to assess muscle changes in animals exposed to two new models: hindlimb suspension using a pelvic harness (HLS) and a partial weight-bearing (PWB) model that mimics partial gravity (including Lunar and Martian gravities). We also used a simple needle array electrode in lieu of surface or  EIM approaches previously employed. Our HLS results confirmed earlier findings obtained after spaceflight and tail suspension. Indeed, one EIM measure (i.e., phase-slope) that was previously reported as highly sensitive, was significantly decreased after HLS (day 0: 14.60 ± 0.97, day 7: 11.03 ± 0.81, and day 14: 10.13 ± 0.55 | Deg/MHz|,  < 0.0001), and was associated with a significant decrease in muscle grip force. Although EIM parameters such as 50 kHz phase, reactance, and resistance remained variable over 14 days in PWB animals, we identified major PWB-dependent effects at 7 days. Moreover, the data at both 7 and 14 days correlated to previously observed changes in rear paw grip force using the same PWB model. In conclusion, our data suggest that EIM has the potential to serve as biomarker of muscle deconditioning during exposure to both micro- and partial- gravity during future human space exploration.

Janice A. Nagy

Department of Neurology

Harvard Medical School - Beth Israel Deaconess Medical Center

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Janice A. Nagy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology

Harvard Medical School - Beth Israel Deaconess Medical Center

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Department of Neurology