Hemorrhagic shock (HS) is a severe life-threatening condition characterized by loss of blood volume and a lack of oxygen (O₂) delivery to tissues. The objective of this study was to examine the impact of manipulating Starling forces in the microcirculation during HS to increase microvascular perfusion without restoring blood volume or increasing O₂ carrying capacity. To decrease interstitial tissue pressure, we developed a non-contact system to locally apply negative pressure and manipulate the pressure balance in capillaries, while allowing for visualization of the microcirculation. Golden Syrian hamsters were instrumented with dorsal window chambers and subjected to a controlled hemorrhaged of 50% of the animal's blood volume without any fluid resuscitation. A negative pressure chamber was attached to the dorsal window chamber and a constant negative pressure was applied. Hemodynamic parameters (including microvascular diameter, blood flow, and functional capillary density [FCD]) were measured before and during the four hours following the hemorrhage, with and without applied negative pressure. Blood flow significantly increased in arterioles during negative pressure. The increase in flow through arterioles also improved microvascular perfusion as reflected by increased FCD. These results indicate that negative pressure increases flow in the microcirculation when fluid resuscitation is not available, thus restoring blood flow, oxygen delivery, and preventing the accumulation of metabolic waste. Applying negative pressure might allow for control of microvascular blood flow and oxygen delivery to specific tissue areas.

The microcirculation is a complex network of vessels ranging from as large as 100 μm to as small as 5 μm. This complex network is responsible for the regulation of oxygen to the surrounding tissues and ensures metabolite washout. With a more complete understanding of the microcirculation's physiologic and pathologic tendencies, engineers can create new solutions to combat blood pathologies and shock-related diseases. Over the last number of decades a grown interest in the microcirculation has resulted in the development of fundamental techniques to quantify the microvasculature flow and the release of oxygen to tissues.

Microvascular fluid exchange is primarily dependent on Starling forces and both the active and passive myogenic response of arterioles and post-capillary venules. Arterioles are classically considered resistance vessels, while venules are considered capacitance vessels with high distensibility and low tonic sympathetic stimulation at rest. However, few studies have investigated the effects of modulating interstitial hydrostatic pressure, particularly in the context of hemorrhagic shock. The objective of this study was to investigate the mechanics of arterioles and functional capillary density (FCD) during application of negative tissue interstitial pressure after 40% total blood volume hemorrhagic shock. In this study, we characterized systemic and microcirculatory hemodynamic parameters, including FCD, in hamsters instrumented with a dorsal window chamber and a custom-designed negative pressure application device via intravital microscopy. In large arterioles, application of negative pressure after hemorrhagic shock resulted in a 13 ± 11% decrease in flow compared with only a 7 ± 9% decrease in flow after hemorrhagic shock alone after 90 minutes. In post-capillary venules, however, application of negative pressure after hemorrhagic shock resulted in a 31 ± 4% decrease in flow compared with only an 8 ± 5% decrease in flow after hemorrhagic shock alone after 90 minutes. Normalized FCD was observed to significantly improve after application of negative pressure after hemorrhagic shock (0.66 ± 0.02) compared to hemorrhagic shock without application of negative pressure (0.50 ± 0.04). Our study demonstrates that application of negative pressure acutely improves FCD during hemorrhagic shock, though it does not normalize FCD. These results suggest that by increasing the hydrostatic pressure gradient between the microvasculature and interstitium, microvascular perfusion can be transiently restored in the absence of volume resuscitation. This study has significant clinical implications, particularly in negative pressure wound therapy, and offers an alternative mechanism to improve microvascular perfusion during hypovolemic shock.

Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e. erythrocruorins) derived from annelid sources. Therefore, the goal of this study was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.

Polymerized hemoglobin (PolyHb) is a promising hemoglobin (Hb)-based oxygen carrier currently undergoing development as a red blood cell substitute. Unfortunately, commercially developed products are composed of low-molecular-weight (LMW) PolyHb molecules, which extravasate, scavenge nitric oxide, and result in vasoconstriction and hypertension. The naturally occurring Hb-scavenging species haptoglobin (Hp), combined with the purified heme-scavenging species apohemoglobin (apoHb), is a potential candidate to alleviate the pressor effect of PolyHb. This study evaluated the protective activity of administering the apoHb-Hp complex to mitigate the vasoactive response induced by the transfusion of LMW PolyHb. Hp binding to PolyHb was characterized in vitro. The effectiveness of apoHb-Hp administration on reducing the vasoconstriction and pressor effects of PolyHb was assessed by measuring systemic and microcirculatory hemodynamics. Transfusion of LMW PolyHb to vehicle control pretreated animals increased mean arterial pressure while decreasing arteriole diameter and functional capillary density. However, transfusion of LMW PolyHb to apoHb-Hp pretreated animals prevented changes in mean arterial pressure, heart rate, arteriole diameter, blood flow, and functional capillary density relative to before transfusion. These results indicate that the increased size of PolyHb after binding to the apoHb-Hp complex may help compartmentalize PolyHb in the vascular space and thus reduce extravasation, nitric oxide scavenging, and toxicity responsible for vasoconstriction and systemic hypertension.

Infection with COVID-19 has resulted in over 276,000 deaths in the United States and over 1.5 million deaths globally, with upwards of 15% of patients requiring hospitalization. Severe COVID-19 infection is, in essence, a microvascular disease. This contention has been emphasized throughout the course of the pandemic, particularly due to the clinical manifestation of severe infection. In fact, it has been hypothesized and shown in particular instances that microvascular function is a significant prognosticator for morbidity and mortality. Initially thought to be isolated to the pulmonary system and resulting in ARDS, patients with COVID-19 have been observed to have acute cardiac, renal, and thrombolytic complications. Therefore, severe COVID-19 is a vascular disease that has systemic implications. The objective of this review is to provide a mechanistic background for the microvascular nature of severe COVID-19 infection, with a particular emphasis on dysfunction of the endothelial glycocalyx and nitric oxide mediated pathogenesis.

Cisplatin is a promising therapeutic for the treatment of non-small cell lung cancer (NSCLC). Unfortunately, a significant portion of NSCLC patients relapse due to cisplatin chemoresistance. This chemoresistance is thought to be primarily associated with hypoxia in the tumor microenvironment. Administration of hemoglobin (Hb)-based oxygen (O₂) carriers (HBOCs) is a promising strategy to alleviate hypoxia in the tumor, which may make cisplatin more effective. In this study, we administered a high O₂ affinity, relaxed state (R-state) polymerized hemoglobin (PolyHb) to three different NSCLC cell lines cultured in vitro and implanted in vivo into healthy mice. The R-state PolyHb administered in this study is unable to deliver O₂ unless under severe hypoxia which significantly limits its oxygenation potential. In vitro sensitivity studies indicate that the administration of PolyHb increases the effectiveness of cisplatin under hypoxic conditions. Additional animal studies revealed that co-administration of PolyHb with cisplatin attenuated tumor growth without alleviating hypoxia. Analysis of reactive O₂ species production in the presence of hypoxic culture indicates that exogenous ROS production by oxidized PolyHb may the mechanism of chemosensitization. This ROS mechanism, coupled with oxygenation, may be a potential chemosensitizing strategy for use in NSCLC treatment.

Microcirculatory preservation is essential for patient recovery from hemorrhagic shock. In hemorrhagic shock, microcirculatory flow and pressure are greatly reduced, creating an oxygen debt that may eventually become irreversible. During shock, tissues become hypoxic, cellular respiration turns to anaerobic metabolism, and the microcirculation rapidly begins to fail. This condition requires immediate fluid resuscitation to promote tissue reperfusion. The choice of fluid for resuscitation is whole blood; however, this may not be readily available and, on a larger scale, may be globally insufficient. Thus, extensive research on viable alternatives to blood has been undertaken in an effort to develop a clinically deployable blood substitute. This has not, as of yet, achieved fruition, in part due to an incomplete understanding of the complexities of the function of blood in the microcirculation. Hemodynamic resuscitation is acknowledged to be contingent on a number of factors other than volume expansion. The circulation of whole blood is carefully regulated to optimize oxygen delivery to the tissues via shear stress modulation through blood viscosity, inherent oxygen-carrying capacity, cell-free layer variation, and myogenic response, among other variables. Although plasma expanders can address a number of these issues, hemoglobin-based oxygen carriers (HBOCs) introduce a method of replenishing the intrinsic oxygen-carrying capacity of blood. There continue to be a number of issues related to HBOCs, but recent advances in the next-generation HBOCs show promise in the preservation of microcirculatory function and limiting toxicities. The development of HBOCs is now focused on viscosity and the degree of microvascular shear stress achieved in order to optimize vasoactive and oxygen delivery responses by leveraging the restoration and maintenance of physiological responses to blood flow in the microcirculation. Blood substitutes with higher viscous properties tend to improve oxygen delivery compared to those with lower viscosities. This review details current concepts in blood substitutes, particularly as they relate to trauma/hemorrhagic shock, with a specific focus on their complex interactions in the microcirculation.