Dilation of the superior sagittal sinus detected in rat model of mild traumatic brain injury using 1 T magnetic resonance imaging.

Introduction: Mild traumatic brain injury (mTBI) is a common injury that can lead to temporary and, in some cases, life-long disability. Magnetic resonance imaging (MRI) is widely used to diagnose and study brain injuries and diseases, yet mTBI remains notoriously difficult to detect in structural MRI. mTBI is thought to be caused by microstructural or physiological changes in the function of the brain that cannot be adequately captured in structural imaging of the gray and white matter. However, structural MRIs may be useful in detecting significant changes in the cerebral vascular system (e.g., the blood-brain barrier (BBB), major blood vessels, and sinuses) and the ventricular system, and these changes may even be detectable in images taken by low magnetic field strength MRI scanners (<1.5T). Methods: In this study, we induced a model of mTBI in the anesthetized rat animal model using a commonly used linear acceleration drop-weight technique. Using a 1T MRI scanner, the brain of the rat was imaged, without and with contrast, before and after mTBI on post-injury days 1, 2, 7, and 14 (i.e., P1, P2, P7, and P14). Results: Voxel-based analyses of MRIs showed time-dependent, statistically significant T2-weighted signal hypointensities in the superior sagittal sinus (SSS) and hyperintensities of the gadolinium-enhanced T1-weighted signal in the superior subarachnoid space (SA) and blood vessels near the dorsal third ventricle. These results showed a widening, or vasodilation, of the SSS on P1 and of the SA on P1-2 on the dorsal surface of the cortex near the site of the drop-weight impact. The results also showed vasodilation of vasculature near the dorsal third ventricle and basal forebrain on P1-7. Discussion: Vasodilation of the SSS and SA near the site of impact could be explained by the direct mechanical injury resulting in local changes in tissue function, oxygenation, inflammation, and blood flow dynamics. Our results agreed with literature and show that the 1T MRI scanner performs at a level comparable to higher field strength scanners for this type of research.

Prolonged Electro-muscular Incapacitation in a Porcine Model Causes Spinal Injury.

Conducted electrical weapons are designed to cause temporary electro-muscular incapacitation (EMI) without significant injury. The objective of this study was to assess the risk and cause of spinal injury due to exposure to a benchtop EMI device. Porcine subjects were exposed to 19 and 40 Hz electrical stimuli for a prolonged duration of 30 sec. X-ray imaging, necropsy, and accelerometry found that lumbosacral spinal fractures occurred in at least 89% of all subjects, regardless of the stimulus group, and were likely caused by musculoskeletal fatigue-related stress in the lumbosacral spine. Spinal fractures occurred in the porcine model at an unusually high rate compared to human. This may be due to both the prolonged duration of electrical stimulation and significant musculoskeletal differences between humans and pigs, which suggests that the porcine model is not a good model of EMI-induced spinal fracture in humans.

Increased Hematocrit Due to Electrical-Waveform Exposures in Splenectomized Sus scrofa.

In laboratory studies of the pig Sus scrofa, hematocrit has consistently increased after conducted-electrical-weapon (CEW) exposures, possibly due to contraction of the spleen. Splenectomized animals and intact sham control animals were exposed, each for 30 sec, to a benchtop-produced electrical waveform of net charge levels similar to those of some CEWs. Changes in the blood were compared statistically. Hematocrit increased significantly in both splenectomized and sham animals. There were no significant main-effect differences between values of hematocrit from the two groups. There were, however, significant interactive effects of time and splenectomy for hematocrit, red blood cell count, and hemoglobin. After peak values were reached for these variables, values returned toward baseline levels more slowly in splenectomized animals. This may have been due to the lack of a spleen to sequester red blood cells (thereby resulting in more cells remaining in the general circulation), unlike sham animals with intact spleens.

Deterioration of red blood cell mechanical properties is reduced in anaerobic storage.

BACKGROUND: Hypothermic storage of red blood cells (RBCs) results in progressive deterioration of the rheological properties of the cells, which may reduce the efficacy of RBC transfusions. Recent studies have suggested that storing RBC units under anaerobic conditions may reduce this storage-induced deterioration. MATERIALS AND METHODS: The aim of this study was to compare the rheological properties of conventionally and anaerobically stored RBC and provide a measure of the relationship between oxidative damage to stored RBC and their ability to perfuse microvascular networks. Three different microfluidic devices were used to measure the ability of both types of stored RBC to perfuse artificial microvascular networks. Flow rates of the RBC passing through the entire network (bulk perfusion) and the individual capillaries (capillary perfusion) of the devices were measured on days 2, 21, 42, and 63 of storage. RESULTS: The bulk perfusion rates for anaerobically stored RBC were significantly higher than for conventionally stored RBCs over the entire duration of storage for all devices (up to 10% on day 42; up to 14% on day 63). Capillary perfusion rates suggested that anaerobically stored RBC units contained significantly fewer non-deformable RBC capable of transiently plugging microfluidic device capillaries. The number of plugging events caused by these non-deformable RBC increased over the 63 days of hypothermic storage by nearly 16- to 21-fold for conventionally stored units, and by only about 3- to 6-fold for anaerobically stored units. DISCUSSION: The perfusion measurements suggest that anaerobically stored RBC retain a greater ability to perfuse networks of artificial capillaries compared to conventionally (aerobically) stored RBC. It is likely that anaerobic storage confers this positive effect on the bulk mechanical properties of stored RBC by significantly reducing the number of non-deformable cells present in the overall population of relatively well-preserved RBC.

When physics meets biology: low and high-velocity penetration, blunt impact, and blast injuries to the brain.

The incidence of traumatic brain injuries (TBI) in the US has reached epidemic proportions with well over 2 million new cases reported each year. TBI can occur in both civilians and warfighters, with head injuries occurring in both combat and non-combat situations from a variety of threats, including ballistic penetration, acceleration, blunt impact, and blast. Most generally, TBI is a condition in which physical loads exceed the capacity of brain tissues to absorb without injury. More specifically, TBI results when sufficient external force is applied to the head and is subsequently converted into stresses that must be absorbed or redirected by protective equipment. If the stresses are not sufficiently absorbed or redirected, they will lead to damage of extracranial soft tissue and the skull. Complex interactions and kinematics of the head, neck and jaw cause strains within the brain tissue, resulting in structural, anatomical damage that is characteristic of the inciting insult. This mechanical trauma then initiates a neuro-chemical cascade that leads to the functional consequences of TBI, such as cognitive impairment. To fully understand the mechanisms by which TBI occurs, it is critically important to understand the effects of the loading environments created by these threats. In the following, a review is made of the pertinent complex loading conditions and how these loads cause injury. Also discussed are injury thresholds and gaps in knowledge, both of which are needed to design improved protective systems.

Biophysical mechanisms of traumatic brain injuries.

Despite years of effort to prevent traumatic brain injuries (TBIs), the occurrence of TBI in the United States alone has reached epidemic proportions. When an external force is applied to the head, it is converted into stresses that must be absorbed into the brain or redirected by a helmet or other protective equipment. Complex interactions of the head, neck, and jaw kinematics result in strains in the brain. Even relatively mild mechanical trauma to these tissues can initiate a neurochemical cascade that leads to TBI. Civilians and warfighters can experience head injuries in both combat and noncombat situations from a variety of threats, including ballistic and blunt impact, acceleration, and blast. It is critical to understand the physics created by these threats to develop meaningful improvements to clinical care, injury prevention, and mitigation. Here the authors review the current state of understanding of the complex loading conditions that lead to TBI and characterize how these loads are transmitted through soft tissue, the skull and into the brain, resulting in TBI. In addition, gaps in knowledge and injury thresholds are reviewed, as these must be addressed to better design strategies that reduce TBI incidence and severity.

Spontaneous oscillations of capillary blood flow in artificial microvascular networks.

Previous computational studies have suggested that the capillary blood flow oscillations frequently observed in vivo can originate spontaneously from the non-linear rheological properties of blood, without any regulatory input. Testing this hypothesis definitively in experiments involving real microvasculature has been difficult because in vivo the blood flow in capillaries is always actively controlled by the host. The objective of this study was to test the hypothesis experimentally and to investigate the relative contribution of different blood cells to the capillary blood flow dynamics under static boundary conditions and in complete isolation from the active regulatory mechanisms mediated by the blood vessels in vivo. To accomplish this objective, we passed whole blood and re-constituted blood samples (purified red blood cells suspended in buffer or in autologous plasma) through an artificial microvascular network (AMVN) comprising completely inert, microfabricated vessels with the architecture inspired by the real microvasculature. We found that the flow of blood in capillaries of the AMVN indeed oscillates with characteristic frequencies in the range of 0-0.6 Hz, which is in a very good agreement with previous computational studies and in vivo observations. We also found that the traffic of leukocytes through the network (typically neglected in computational modeling) plays an important role in generating the oscillations. This study represents the key piece of experimental evidence in support of the hypothesis that spontaneous, self-sustained oscillations of capillary blood flow can be generated solely by the non-linear rheological properties of blood flowing through microvascular networks, and provides an insight into the mechanism of this fundamentally important microcirculatory phenomenon.

Artificial microvascular network: a new tool for measuring rheologic properties of stored red blood cells.

BACKGROUND: The progressive deterioration of red blood cell (RBC) rheologic properties during refrigerated storage may reduce the clinical efficacy of transfusion of older units. STUDY DESIGN AND METHODS: This article describes the development of a microfluidic device designed to test the rheologic properties of stored RBCs by measuring their ability to perfuse an artificial microvascular network (AMVN) comprised of capillary-size microchannels arranged in a pattern inspired by the real microvasculature. In the AMVN device, the properties of RBCs are evaluated by passing a 40% hematocrit suspension of RBCs through the network and measuring the overall perfusion rate. RESULTS: The sensitivity of the AMVN device to the storage-induced change in rheologic properties of RBCs was tested using five prestorage leukoreduced RBC units stored in AS-1 for 41 days. The AMVN perfusion rate for stored RBCs was 26 ± 4% (19%-30%) lower than for fresh RBCs. Washing these stored RBCs in saline improved their performance by 41 ± 6% (the AMVN perfusion rate for washed stored RBCs was still 15 ± 2% lower than for fresh RBCs). CONCLUSIONS: The measurements performed using the AMVN device confirm a significant decline in the rheologic properties of RBCs in units nearing expiration and demonstrate the sensitivity of the device to these storage-induced changes. The AMVN device may be useful for testing the effect of new storage conditions, additive solutions, and rejuvenation strategies on the rheologic properties of stored RBCs in vitro.

Traffic of leukocytes in microfluidic channels with rectangular and rounded cross-sections.

Traffic of leukocytes in microvascular networks (particularly through arteriolar bifurcations and venular convergences) affects the dynamics of capillary blood flow, initiation of leukocyte adhesion during inflammation, and localization and development of atherosclerotic plaques in vivo. Recently, a growing research effort has been focused on fabricating microvascular networks comprising artificial vessels with more realistic, rounded cross-sections. This paper investigated the impact of the cross-sectional geometry of microchannels on the traffic of leukocytes flowing with human whole blood through a non-symmetrical bifurcation that consisted of a 50 µm mother channel bifurcating into 30 µm and 50 µm daughter branches. Two versions of the same bifurcation comprising microchannels with rectangular and rounded cross-sections were fabricated using conventional multi-layer photolithography to produce rectangular microchannles that were then rounded in situ using a recently developed method of liquid PDMS/air bubble injection. For microchannels with rounded cross-sections, about two-thirds of marginated leukocytes traveling along a path in the top plane of the bifurcation entered the smallest 30 µm daughter branch. This distribution was reversed in microchannels with rectangular cross-sections--the majority of leukocytes traveling along a similar path continued to follow the 50 µm microchannels after the bifurcation. This dramatic difference in the distribution of leukocyte traffic among the branches of the bifurcation can be explained by preferential margination of leukocytes towards the corners of the 50 µm mother microchannels with rectangular cross-sections, and by the additional hindrance to leukocyte entry created by the sharp transition from the 50 µm mother microchannel to the 30 µm daughter branch at the intersection. The results of this study suggest that the trajectories of marginated leukocytes passing through non-symmetrical bifurcations are significantly affected by the cross-sectional geometry of microchannels and emphasize the importance of using microfludic systems with geometrical configurations closely matching physiological configurations when modeling the dynamics of whole blood flow in the microcirculation.

Passive recruitment of circulating leukocytes into capillary sprouts from existing capillaries in a microfluidic system.

Recent evidence implicating leukocytes in angiogenesis raises the question of whether leukocytes and other cells circulating with the blood in microvascular networks can home to capillary sprouts intraluminally. This study describes an investigation of leukocyte trafficking in sprouting capillaries fabricated using soft lithography. The leukocytes passing with whole blood through existing capillaries were able to enter microfabricated capillary sprouts of variable length and sprouting angle due to the mechanical interaction with red blood cells (RBCs) at the sprouting bifurcation, in spite of the complete absence of blood flow through the blind-ended sprouts or any chemoattractants. The RBCs formed "comet tails" (the densely packed cellular trains forming behind leukocytes as they move through narrow capillaries) and effectively pushed leukocytes into the microfabricated sprouts while bypassing them at the sprouting bifurcation. Individual sprouts filled with several leukocytes, as wells as RBCs and platelets, were observed. The results of this study suggest that (i) blood cells are likely present in capillary sprouts throughout their development, (ii) leukocytes and other circulating cells may use this mechanism to home to capillary sprouts intraluminally for direct engraftment, and (iii) tissues may use this phenomenon as another mechanism for local recruitment of leukocytes from the blood stream.

Systemic lupus erythematosus serum deposits C4d on red blood cells, decreases red blood cell membrane deformability, and promotes nitric oxide production.

OBJECTIVE: Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBCs). The aim of this study was to address whether this process affects the biophysical properties of RBCs. METHODS: Serum and RBCs were isolated from patients with SLE and healthy controls. RBCs from healthy universal donors (type O, Rh negative) were incubated with SLE or control serum. We used flow cytometry to assess complement fragment deposition on RBCs. RBC membrane deformability was measured using 2-dimensional microchannel arrays. Protein phosphorylation levels were quantified by Western blotting. RESULTS: Incubation of healthy universal donor RBCs with sera from patients with SLE, but not with control sera, led to deposition of C4d fragments on the RBCs. Complement-decorated RBCs exhibited significant decreases in both membrane deformability and flickering. Sera from SLE patients triggered a transitory Ca(++) influx in RBCs that was associated with decreased phosphorylation of ß-spectrin and with increased phosphorylation of band 3, two key proteins of RBC cytoskeleton. Finally, incubation with SLE sera led to the production of nitric oxide by RBCs, whereas this did not occur with control sera. CONCLUSION: Our data suggest that complement activation in patients with SLE leads to calcium-dependent cytosketeletal changes in RBCs that render them less deformable, probably impairing their flow through capillaries. This phenomenon may negatively affect the delivery of oxygen to the tissues.

Ligation of complement receptor 1 increases erythrocyte membrane deformability.

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca(++) influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca(++) influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of ß-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.