2. A normal human contains 5 L of blood, approximately 2% of which is resident i
ID: 28843 • Letter: 2
Question
2. A normal human contains 5 L of blood, approximately 2% of which is resident in the systemic (i.e., non-pulmonary) capillaries at any given time. (1) Assuming that the capillaries are 8 ?m in diameter, estimate the total length of capillaries in the body (excluding the lungs). (2) If an average capillary length is 1 mm, how many capillaries are there in the body? (3) Cardiac output is 5 L/min. Assume that this is evenly distributed throughout a parallel network consisting of the capillaries found in (2), estimate the pressure drop across the capillary bed. Assume Newtonian, laminar flow in the capillaries with ?eff of 3.5 cP. What percentage of the total 85 mmHg systemic pressure drop is this?Explanation / Answer
Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis. Hemoglobin (Hb)–induced neurotoxicity is a major pathophysiological feature of intracerebral (ICH) and subarachnoid hemorrhage.1–3 The lysis of extravasated red blood cells exposes the central nervous system (CNS) to significant quantities of Hb or its breakdown products, triggering harmful oxidative and inflammatory events.1–3 Commonly studied experimental models of ICH and subarachnoid hemorrhage include cerebral vessel perforation, collagenase digestion, or the direct injection of Hb or blood into brain compartments outside the confines of the blood-brain barrier (BBB).3–7 Given that an intact BBB blocks CNS entry of extracellular Hb, considerably less attention has focused on studying the potential CNS effects of plasma cell-free Hb. For example, relatively little is known about how systemic exposure to Hb may affect the function and integrity of the BBB. This may be particularly important given the well-documented susceptibility of vascular endothelium to Hb or heme-mediated oxidative injury and the reported vasculopathies associated with chronic or intermittent hemolysis.8–12 The development of oxygen-carrying solutions that use cell-free Hb as a basis for chemical modification has been linked to serious adverse events, including stroke and myocardial infarction, in late-phase clinical trials.13 The mechanisms underlying these events are not fully understood but could be related, in part, to the uncontrolled pro-oxidative activity of cell-free Hb or heme.8,9 Similarly, neurologic complications (eg, stroke) are commonly observed in disease states associated with chronic or intermittent intravascular hemolysis, including genetic and drug-induced hemolytic anemias, microbial infections, and trauma.11,12,14–18 However, the mechanisms are multifaceted and not fully defined.12,17,18 Extracellular Hb can oxidize to methemoglobin (ferric, Fe3+), ferryl heme intermediate (Fe4+), hemichromes, and free heme or iron, which can initiate or propagate oxidative damage to lipids, nucleic acids, and proteins.8,9 Cell-free Hb is also an effective scavenger of nitric oxide, a key mediator of vascular homeostasis.11,14 Heme catabolism and iron sequestration systems play a key role in mediating tissue responses to cell-free Hb.8,9,19 Heme oxygenase (HO), the rate-limiting enzyme of heme catabolism, exists as two main isoforms, inducible HO-1 and constitutive HO-2 that catalyze the degradation of heme to biliverdin, free iron, and carbon monoxide.20 HO-1 is up-regulated by heme, heavy metals, lipopolysaccharide, and several other stimuli. In the brain, HO-1 induction has been reported in ICH, subarachnoid hemorrhage, and other neurodegenerative conditions.7,21–26 HO-1 up-regulation is viewed as an adaptive response that confers protection through anti-inflammatory, anti-apoptotic, and antioxidant mechanisms.20,24,25 However, under settings of excess heme exposure, HO-1 over-activation may lead to iron or other by-product toxicities and can serve as a marker of oxidative stress.7,23,26,27 The BBB is composed of endothelial cells, basal lamina, astrocytic end-foot processes, pericytes, perivascular macrophages, and neurons.28,29 Tight junctions (TJs) are specialized interendothelial structural complexes that restrict paracellular permeability. TJs are composed of transmembrane proteins, including occludin, claudins (primarily claudin-3 and -5), junctional adhesion molecules (JAMs), and cytoplasmic zonula occludens (ZO) family members (ZO-1, -2, -3). ZO proteins are accessory proteins that bind the other proteins to the actin cytoskeleton.28,29 Astrocytes, pericytes, and perivascular macrophages play a central role in BBB regulation. Activated astrocytes express increased levels of glial fibrillary acidic protein (GFAP), which serves as a reliable marker of BBB dysfunction or injury.30,31 Pericytes are enveloped within the basal lamina and provide direct structural support to the cerebral endothelium as well as metabolic support through the release of growth factors.32,33 Perivascular macrophages are located abluminally and serve as phagocytic regulators of the perivascular environment.32 In this study, we examined the effects of a polymerized cell-free Hb on BBB integrity and oxidative stress in a guinea pig exchange transfusion model. This model was selected on the basis of previous studies that identified the guinea pig as a potentially useful species for studying the oxidative toxicity of cell-free Hb.9,19,34 The guinea pig, similar to humans, lacks the ability to produce ascorbate, which is a powerful reductant that minimizes the oxidation of cell-free Hb in the circulation.9,34 Here, we provide evidence that systemic Hb exposure triggers BBB disruption and oxidative stress in this model. Go to: Materials and Methods Materials Polymerized bovine hemoglobin (Oxyglobin; HbG), a product approved for veterinary use, was a kind gift from Biopure Corporation (Cambridge, MA). This solution consists of a heterogeneous mixture of glutaraldehyde-polymerized bovine hemoglobin at a concentration of 13 g/dl in modified Lactated Ringer's solution. The solution contains unstabilized tetramers (Related Questions
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