Effect of oxygen affinity on systemic perfusion and brain tissue oxygen tension after extreme hemodilution with hemoglobin-starch conjugates in rats

Purpose: To determine the oxygen affinity for optimal tissue oxygen delivery with a hemoglobin-hydroxyethyl starch conjugate (HRC 101). Methods: Anesthetized rats were hemodiluted (180 ml kg -1) with low (P 50 ∼ 70 mmHg) or high affinity (P 50 ∼ 14 mmHg) HRC 101 at hemoglobin (Hb) concentrations near 100 or 70 g l -1 (n = 6-8). Hippocampal tissue oxygen tension (P BrO 2), blood flow, arterial blood gases, Hb, hematocrit (Hct) and lactate were measured. Data (mean ± SD) were analyzed by two-way ANOVA. Results: Hemodilution reduced the hematocrit to 1 ± 1% in all groups. PB rO 2 was best maintained after hemodilution with low affinity HRC 101 at Hb 100 and 70 g l -1(25.2 ± 7.6 and 16.6 ± 8.3 torr, respectively). P BrO 2 decreased (9.5 ± 9.3 torr, P < 0.05) and serum lactate levels increased (5.0 ±1.7 mmol l -1, P < 0.05) following hemodilution with the high affinity HRC 101 (Hb 100 g l -1). Conclusions: HRC 101 with a lower oxygen affinity favored tissue perfusion and maintained P BrO 2 after near complete blood volume exchange in rats. © Springer-Verlag 2009.link_to_subscribed_fulltex

A potential role for inducible nitric oxide synthase in the cerebral response to acute hemodilution

Purpose: Acute anemia increases the cerebral expression of hypoxic molecules including neuronal nitric oxide synthase (nNOS) and hypoxia inducible factor-1α (HIF-1α). This study assessed the effects of acute hemodilution on inducible NOS (iNOS) and systemic inflammatory cytokines. Methods: Anesthetized rats (n = 5-7 per group) underwent 50% hemodilution with pentastarch, whole blood exchange or no fluid exchange. Cerebral cortical nNOS and iNOS protein levels were characterized using Western blot analysis and immunostaining (1 and 18 h). Plasma cytokine levels were assessed by enzyme-linked immunosorbent assay (1, 4, and 18 h). Data were analyzed by two-way analysis of variance to determine significance (P < 0.05, mean ± SD). Results: No differences in mean arterial blood pressure or arterial blood gases were observed between groups after hemodilution. A comparable hemoglobin target (~70 g • L-1) was achieved in all groups following hemodilution. Cerebral cortical iNOS protein levels were increased in anemic rats, relative to controls. The nNOS protein levels increased to a greater degree (P < 0.05 for both). Immunostaining demonstrated that iNOS localized to endothelium, glial fibrillary acidic protein (GFAP) positive (astrocytes) and GFAP negative cells within the brain. Plasma cytokine levels (tumour necrosis factor α, interleukin (IL)-1β and IL-6) increased transiently, to the same levels, in both control and hemodiluted rats. Conclusions: Cerebral cortical iNOS and nNOS protein levels were both increased in anemic rats. The nNOS response was predominant. This suggests that NOS-derived NO may be an important signalling pathway which is activated in the brain during anemia. These cellular responses could maintain cerebral homeostasis, or contribute to neuronal injury, during acute hemodilutional anemia. © 2009 Canadian Anesthesiologists' Society.link_to_subscribed_fulltex

Effect of oxygen affinity and molecular weight of HBOCs on cerebral oxygenation and blood pressure in rats

Purpose: This study assessed the effect of oxygen affinity and molecular weight (MW) of o-raffinose cross-linked hemoglobin based oxygen carriers (HBOCs) on cerebral oxygen delivery and mean arterial blood pressure (MAP) following hemorrhage and resuscitation in rats. Methods: Isoflurane anesthetized rats (n = 6-7 per group) underwent 30% hemorrhage and resuscitation with an equivalent volume of one of three different HBOCs: I) High P50 Poly o-raffinose hemoglobin (Poly OR-Hb, P50 = 70 mmHg); 2) High P50 > 128 Poly OR-Hb (MW > 128 kDa, P50 = 70 mmHg) and 3) Low P50 > 128 Poly OR-Hb (MW > 128 kDa, P50 = 11 mmHg). Hippocampal cerebral tissue oxygen tension, regional cerebral blood flow (rCBF), MAP total hemoglobin concentration and arterial blood gases were measured. Data analysis by two-way ANOVA and post hoc Tukey tests determined significance (P 128 kDa) HBOC group (119 ± 15 mmHg or 127 ± 18 respectively, P < 0.05 for both). Conclusions: O-raffinose polymerized HBOC, with or without lower MW components, maintained cerebral tissue oxygen delivery following hemorrhage and resuscitation in rats. The higher MW HBOCs showed a decrease in peak MAP, which did not alter oxygen delivery. No significant effect of oxygen affinity on cerebral tissue oxygen tension or blood flow was observed.link_to_subscribed_fulltex

Effects of resuscitation fluid on neurologic physiology after cerebral trauma and hemorrhage

BACKGROUND: The current standard of care for fluid resuscitation of hemorrhagic hypotensive patients involves the use of crystalloid solutions. Traumatic brain injury (TBI) is often associated with hemorrhage and hypotension, which can contribute significantly to morbidity and mortality. Guidelines for the choice of fluid resuscitation and the use of red blood cell transfusions are not yet clear in the context of brain injury. METHODS: Various fluid resuscitation strategies were evaluated in Sprague-Dawley rats using fresh blood, normal saline, hypertonic saline, and albumin fluid resuscitation protocols. Mean arterial blood pressure (MAP) and cerebral oximetry were assessed in hemorrhaged groups and the mean population spike amplitudes (PSA) from the hippocampus were examined in fluid percussion injured (FPI) animals subject to hemorrhage and fluid resuscitation. RESULTS: MAP in control animals, hemorrhage and hemorrhage + albumin treated groups was 82.4 ± 1.5 mm Hg, 55.7 ± 1.5 mm Hg, and 97.0 ± 3.4 mm Hg, respectively. Arterial PaO2 was higher in albumin-treated animals relative to other fluid alternatives. Regional tissue oxygen tension (PbrO2) levels in hemorrhaged animals reached significantly higher levels in albumin treated group compared with in normal saline and hypertonic saline (p < 0.001, p = 0.034, respectively). After FPI+hemorrhage, PSA values in albumin- resuscitated animals were significantly higher than in normal saline-resuscitated animals (p = 0.012). CONCLUSIONS: The results of normal saline resuscitation, relative to other fluid alternatives, suggest that a re-evaluation of current treatment strategies in hemorrhagic hypotensive TBI patients is warranted. Albumin demonstrated the greatest beneficial effects on neurophysiology endpoints over crystalloid alternatives. These data suggests that albumin resuscitation may play an important role in the treatment of hemorrhagic hypotension and TBI. © 2008 Lippincott Williams & Wilkins, Inc.link_to_subscribed_fulltex

β2 adrenergic antagonist inhibits cerebral cortical oxygen delivery after severe haemodilution in rats

Background. Haemodilution has been associated with neurological morbidity in surgical patients. This study tests the hypothesis that inhibition of cerebral vasodilatation by systemic β2 adrenergic blockade would impair cerebral oxygen delivery leading to tissue hypoxia in severely haemodiluted rats. Methods. Under general an aesthesia, cerebral tissue probes were placed to measure temperature, regional cerebral blood flow (rCBF) and tissue oxygen tension (PBrO2) in the parietal cerebral cortex or hippocampus. Baseline measurements were established before and after systemic administration of either a β2 antagonist (10 mg kg-1 i.v., ICI 118, 551) or saline vehicle. Acute haemodilution was then performed by simultaneously exchanging 50% of the estimated blood volume (30 ml kg-1) with pentastarch. Arterial blood gases (ABGs), haemoglobin concentration (co-oximetry), mean arterial blood pressure (MAP) and heart rate (HR) were also measured. Data were analysed using a two-way anova and post hoc Tukey's test [mean (sd)]. Results. Haemodilution reduced the haemoglobin concentration comparably in all groups [71 (9) g litre-1]. There were no differences in ABGs, co-oximetry, HR and MAP measurements between control and β2 blocked rats, either before or 60 min after drug or vehicle administration. In rats treated with the β2 antagonist there was a significant reduction in parietal cerebral cortical temperature, regional blood flow and tissue oxygen tension, relative to control rats, 60 min after haemodilution (P<0.05 for each). These differences were not observed when probes were placed in the hippocampus. Conclusion. Systemic β2 adrenergic blockade inhibited the compensatory increase in parietal cerebral cortical oxygen delivery after haemodilution thereby reducing cerebral cortical tissue oxygen tension. © 2006 Oxford University Press.link_to_subscribed_fulltex

Treatment of traumatic brain injury using zinc-finger protein gene therapy targeting VEGF-A

Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery. © Mary Ann Liebert, Inc..link_to_subscribed_fulltex

Reassessing the risk of hemodilutional anemia: Some new pieces to an old puzzle

Purpose: Clinical studies demonstrate that anemia increases the risk of morbidity and mortality. Tissue hypoxia is an attractive but incompletely characterized candidate mechanism of anemia-induced organ injury. Physiological responses that optimize tissue oxygen delivery (nitric oxide synthase-NOS) and promote cellular adaptation to tissue hypoxia (hypoxia inducible factor-HIF) may reduce the risk of hypoxic organ injury and thereby improve survival during anemia. The presence of vascular diseases would likely impair the efficacy of these physiological mechanisms, increasing the risk of anemia-induced organ injury. In all cases, biological signals that indicate the activation of these adaptive mechanisms could provide an early and treatable warning signal of impending anemia-induced organ injury. Thus, we review the evidence for tissue hypoxia during acute hemodilutional anemia and also explore the novel hypothesis that methemoglobin, a measurable byproduct of increased NOS-derived nitric oxide (NO), may serve as a biomarker for "anemic stress". Source: Published peer-reviewed studies provided the main source of information. Data from experimental studies were reassessed to derive the relationship between hemodilution (reduced hemoglobin concentration) and increased methemoglobin levels. Principal findings: Active physiological mechanisms (sympathetic nervous system) are required to maintain optimal tissue oxygen delivery during hemodilutional anemia. Despite these responses, tissue hypoxia occurs during acute hemodilution, as demonstrated by a decrease in tissue PO2 and an increase in hypoxic cellular responses (NOS, HIF). Optimal tissue oxygen delivery may be compromised further when cardiovascular responses are impaired. The positive correlation between decreased hemoglobin concentration (Hb) and an increase in methemoglobin levels in acutely anemic animals supports the hypothesis that anemia-induced increases in tissue NOS activity could promote methemoglobin formation. Methemoglobin may be a measurable byproduct of NO-mediated Hb oxidation. Conclusions: Evidence continues to demonstrate that anemia increases morbidity and mortality, possibly via hypoxic mechanisms. A potential strategy for assessing "anemic stress" was derived from experimental data based on a readily measurable biomarker, methemoglobin. New methods for measuring real-time hemoglobin and methemoglobin levels in patients may provide the basis to translate this idea into clinical practice. Further mechanistic studies are required to determine if the impact of reduced tissue oxygen delivery and activation of hypoxic cellular mechanism can be linked to measurable changes in biomarkers and clinical outcomes in acutely anemic patients. © 2010 Canadian Anesthesiologists' Society.link_to_subscribed_fulltex

Treatment with a highly selective β1 antagonist causes dose-dependent impairment of cerebral perfusion after hemodilution in rats

BACKGROUND: Acute β-blockade has been associated with a dose-dependent increase in adverse outcomes, including stroke and mortality. Acute blood loss contributes to the incidence of these adverse events. In an attempt to link the risks of acute blood loss and β-blockade, animal studies have demonstrated that acute β-blockade impairs cerebral perfusion after hemodilution. We expanded on these fndings by testing the hypothesis that acute β-blockade with a highly β1-specific antagonist (nebivolol) causes dose-dependent cerebral hypoxia during hemodilution. METHODS: Anesthetized rats and mice were randomized to receive vehicle or nebivolol (1.25 or 2.5 mg/kg) IV before hemodilution to a hemoglobin concentration near 60 g/L. Drug levels, heart rate (HR), cardiac output (CO), regional cerebral blood flow (rCBF, laser Doppler), and microvascular brain Po2 (PBrO2, G2 Oxyphor) were measured before and after hemodilution. Endothelial nitric oxide synthase (NOS), neuronal NOS (nNOS), inducible NOS, and hypoxia inducible factor (HIF)-1α were assessed by Western blot. HIF-α expression was also assessed using an HIF-(ODD)-luciferase mouse model. Data were analyzed using analysis of variance with significance assigned at P < 0.05, and corrected P values are reported for all post hoc analyses. RESULTS: Nebivolol treatment resulted in dose-specific plasma drug levels. In vehicle-treated rats, hemodilution increased CO and rCBF (P < 0.010) whereas PBrO 2 decreased to 45.8 ± 18.7 mm Hg (corrected P < 0.001; 95% CI 29.4-69.7). Both nebivolol doses comparably reduced HR and attenuated the CO response to hemodilution (P < 0.012). Low-dose nebivolol did not impair rCBF or further reduce PBrO2 after hemodilution. High-dose nebivolol attenuated the rCBF response to hemodilution and caused a further reduction in PBrO2 to 28.4 ± 9.6 mm Hg (corrected P = 0.019; 95% CI 17.4-42.7). Both nebivolol doses increased brain endothelial NOS protein levels. Brain HIF-1α, inducible NOS, and nNOS protein levels and brain HIF-luciferase activity were increased in the high-dose nebivolol group after hemodilution (P < 0.032). CONCLUSIONS: Our data demonstrate that nebivolol resulted in a dose-dependent decrease in cerebral oxygen delivery after hemodilution as refected by a decrease in brain tissue Po2 and an increase in hypoxic protein responses (HIF-1α and nNOS). Low-dose nebivolol treatment did not result in worsened tissue hypoxia after hemodilution, despite comparable effects on HR and CO. These data support the hypothesis that acute β-blockade with a highly β1-specific antagonist causes a dose-dependent impairment in cerebral perfusion during hemodilution. Copyright © 2013 International Anesthesia Research Society.link_to_subscribed_fulltex

Increased expression of HIF-1α, nNOS, and VEGF in the cerebral cortex of anemic rats

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1α (HIF-1α), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial PaO2 values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 ± 2 g/l. In anemic rats, cerebral cortical HIF-1α protein levels were increased, relative to controls (1.7 ± 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 ± 0.2- and 1.5 ± 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1α and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 ± 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1α protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations. Copyright © 2007 the American Physiological Society.link_to_subscribed_fulltex

Differential HIF and NOS responses to acute anemia: Defining organ-specific hemoglobin thresholds for tissue hypoxia

Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide (NO) synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ-specific Hb thresholds for tissue hypoxia, HIF-α (oxygen-dependent degradation domain, ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50 g/l, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and HIF-2α increased earlier (Hb ~70-90 g/l) in response to anemia. Liver also exhibited an early HIF-α response. Carotid blood flow was increased early (Hb ~70, g/l), but renal blood flow remained relatively constant, only increased at Hb of 50 g/l. Anemia increased nNOS (brain and kidney) and endothelia NOS (eNOS) (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS independent. HIF-dependent RNA levels increased linearly (~10-fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (~100-fold). Plasma EPO levels increased near Hb threshold of 90 g/l, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia. © 2014 the American Physiological Society.link_to_subscribed_fulltex