Elaine Liu

Metoprolol Reduces Cerebral Tissue Oxygen Tension after Acute Hemodilution in Rats

Background:Perioperative &bgr;-blockade and anemia are independent predictors of increased stroke and mortality by undefined mechanisms. This study investigated the effect of &bgr;-blockade on cerebral tissue oxygen delivery in an experimental model of blood loss and fluid resuscitation (hemodilution). Methods:Anesthetized rats were treated with metoprolol (3 mg · kg−1) or saline before undergoing hemodilution with pentastarch (1:1 blood volume exchange, 30 ml · kg−1). Outcomes included cardiac output, cerebral blood flow, and brain (PBrO2) and kidney (PKO2) tissue oxygen tension. Hypoxia inducible factor-1&agr; (HIF-1&agr;) protein levels were assessed by Western blot. Systemic catecholamines, erythropoietin, and angiotensin II levels were measured. Results:Hemodilution increased heart rate, stroke volume, cardiac output (60%), and cerebral blood flow (50%), thereby maintaining PBrO2 despite an approximately 50% reduction in blood oxygen content (P < 0.05 for all). By contrast, PKO2 decreased (50%) under the same conditions (P < 0.05). &bgr;-blockade reduced baseline heart rate (20%) and abolished the compensatory increase in cardiac output after hemodilution (P < 0.05). This attenuated the cerebral blood flow response and reduced PBrO2 (50%), without further decreasing PKO2. Cerebral HIF-1&agr; protein levels were increased in &bgr;-blocked hemodiluted rats relative to hemodiluted controls (P < 0.05). Systemic catecholamine and erythropoietin levels increased comparably after hemodilution in both groups, whereas angiotensin II levels increased only after &bgr;-blockade and hemodilution. Conclusions:Cerebral tissue oxygen tension is preferentially maintained during hemodilution, relative to the kidney, despite elevated systemic catecholamines. Acute &bgr;-blockade impaired the compensatory cardiac output response to hemodilution, resulting in a reduction in cerebral tissue oxygen tension and increased expression of HIF-1&agr;.

Attenuation of the Electrophysiological Function of the Corpus Callosum after Fluid Percussion Injury in the Rat

This study describes a new method used to evaluate axonal physiological dysfunction following fluid percussion induced traumatic brain injury (TBI) that may facilitate the study of the mechanisms and novel therapeutic strategies of posttraumatic diffuse axonal injury (DAI). Stimulated compound action potentials (CAP) were recorded extracellularly in the corpus callosum of superfused brain slices at 3 h, and 1, 3, and 7 days following central fluid percussion injury and demonstrated a temporal pattern of functional deterioration. The maximal CAP amplitude (CAPA) covaried with the intensity of impact 1 day following sham, mild (1.0-1.2 atm), and moderate (1.8-2.0 atm) injury (p < 0.05; 1.11 +/- 0.10, 0.82 +/- 0.11, and 0.49 +/- 0.08 mV, respectively). The CAPA in sham animals were approximately 1.1 mV and did not vary with survival interval (3 h, and 1, 3, and 7 days); however, they were significantly decreased at each time point following moderate injury (p < 0.05; 0.51 +/- 0.11, 0.49 +/- 0.08, 0.46 +/- 0.10, and 0.75 +/- 0.13 mV, respectively). The CAPA at 7 days in the injured group were higher than at 3 h, and 1 and 3 days. H&E and amyloid precursor protein (APP) light microscopic analysis confirmed previously reported trauma-induced axonal injury in the corpus callosum seen after fluid percussion injury. Increased APP expression was confirmed using Western blotting showing significant accumulation at 1 day (IOD 913.0 +/- 252.7; n = 3; p = 0.05), 3 days (IOD 753.1 +/- 159.1; n = 3; p = 0.03), and at 7 days (IOD 1093.8 = 105.0; n = 3; p = 0.001) compared to shams (IOD 217.6 +/- 20.4; n = 3). Thus, we report the characterization of white matter axonal dysfunction in the corpus callosum following TBI. This novel method was easily applied, and the results were consistent and reproducible. The electrophysiological changes were sensitive to the early effects of impact intensity, as well as to delayed changes occurring several days following injury. They also indicated a greater degree of attenuation than predicted by APP expression changes alone.

Heavy neurofilament accumulation and α-spectrin degradation accompany cerebellar white matter functional deficits following forebrain fluid percussion injury

Evidence for diffuse traumatic axonal injury (TAI) in clinical cases and animal models of traumatic brain injury (TBI) indicate that pathophysiological mechanisms extend to regions remote from the injury epicenter. The potential for indirect cerebellar trauma contributing to TBI pathophysiology is of significance since impairment of motor function and coordination is a common consequence of TBI but is also a domain associated with cerebellar function. The relationship between cerebellar white matter structure and function following traumatic head injury has not been examined. Using the fluid percussion injury (FPI) device applied unilaterally in the forebrain, evoked compound action potential (CAP) recordings from cerebellar white matter of Sprague-Dawley rats indicated a spatial and temporal pattern of electrophysiological deficits throughout the cerebellar vermis. The posterior and middle lobules of the cerebellum exhibited significant declines in evoked CAP amplitude compared to sham controls (p=0.004, p=0.005, respectively). Duration of the CAP decay also increased, suggesting that functional white matter deficits were a combination of axonal loss and compromised axonal integrity. Functional white matter deficits persisted at 14 days post-injury in the posterior and middle regions of the cerebellum. Evidence of heavy chain neurofilament (NF200) degradation was observed at 1 day post-injury by Western blot. Immunohistochemistry labeling for NF200 indicated the presence of highly immunoreactive NF200 axonal swellings consistent with morphological features of TAI. alpha-Spectrin degradation was also observed between 1 and 14 days post-injury. This study demonstrates the electrophysiological consequences of cerebellar white matter injury and a temporal profile of NF200 and spectrin degradation following forebrain FPI.

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.

Bone marrow-derived endothelial progenitor cells protect postischemic axons after traumatic brain injury

White matter sparing after traumatic brain injury (TBI) is an important predictor of survival and outcome. Blood vessels and axons are intimately associated anatomically and developmentally. Neural input is required for appropriate vascular patterning, and vascular signaling is important for neuron development and axon growth. Owing to this codependence between endothelial cells and axons during development and the contribution of endothelial progenitor cells (EPCs) in ischemic injury, we hypothesized that EPCs are important in axonal survival after TBI. We examined the effects of allogenic-cultured EPCs on white matter protection and microvascular maintenance after midline fluid percussion injury in adult Sprague–Dawley rats. We used two in vitro models of injury, mechanical stretch and oxygen–glucose deprivation (OGD), to examine the effects of EPCs on the mechanical and ischemic components of brain trauma, respectively. Our results indicate that EPCs improve the white matter integrity and decrease capillary breakdown after injury. Cultured cortical neurons exposed to OGD had less axon degeneration when treated with EPC-conditioned media, whereas no effect was seen in axons injured by mechanical stretch. The results indicate that EPCs are important for the protection of the white matter after trauma and represent a potential avenue for therapy.

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.

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

BACKGROUND:Acute &bgr;-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 &bgr;-blockade, animal studies have demonstrated that acute &bgr;-blockade impairs cerebral perfusion after hemodilution. We expanded on these findings by testing the hypothesis that acute &bgr;-blockade with a highly &bgr;1-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)-1&agr; were assessed by Western blot. HIF-&agr; 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 PBrO2 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-1&agr;, 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 reflected by a decrease in brain tissue PO2 and an increase in hypoxic protein responses (HIF-1&agr; 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 &bgr;-blockade with a highly &bgr;1-specific antagonist causes a dose-dependent impairment in cerebral perfusion during hemodilution.

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 (2.2-2.5 atm) 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 72 h 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.

Metoprolol impairs resistance artery function in mice

Acute β-blockade with metoprolol has been associated with increased mortality by undefined mechanisms. Since metoprolol is a relatively high affinity blocker of β(2)-adrenoreceptors, we hypothesized that some of the increased mortality associated with its use may be due to its abrogation of β(2)-adrenoreceptor-mediated vasodilation of microvessels in different vascular beds. Cardiac output (CO; pressure volume loops), mean arterial pressure (MAP), relative cerebral blood flow (rCBF; laser Doppler), and microvascular brain tissue Po(2) (G2 oxyphor) were measured in anesthetized mice before and after acute treatment with metoprolol (3 mg/kg iv). The vasodilatory dose responses to β-adrenergic agonists (isoproterenol and clenbuterol), and the myogenic response, were assessed in isolated mesenteric resistance arteries (MRAs; ∼200-μm diameter) and posterior cerebral arteries (PCAs ∼150-μm diameter). Data are presented as means ± SE with statistical significance applied at P < 0.05. Metoprolol treatment did not effect MAP but reduced heart rate and stroke volume, CO, rCBF, and brain microvascular Po(2), while concurrently increasing systemic vascular resistance (P < 0.05 for all). In isolated MRAs, metoprolol did not affect basal artery tone or the myogenic response, but it did cause a dose-dependent impairment of isoproterenol- and clenbuterol-induced vasodilation. In isolated PCAs, metoprolol (50 μM) impaired maximal vasodilation in response to isoproterenol. These data support the hypothesis that acute administration of metoprolol can reduce tissue oxygen delivery by impairing the vasodilatory response to β(2)-adrenergic agonists. This mechanism may contribute to the observed increase in mortality associated with acute administration of metoprolol in perioperative patients.

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

PurposeThis 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.MethodsIsoflurane anesthetized rats (n = 6-7 per group) underwent 30% hemorrhage and resuscitation with an equivalent volume of one of three different HBOCs: 1) 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 < 0.05, mean ± SD).ResultsHippocampal tissue oxygen tension increased in all HBOC groups following resuscitation. The rCBF remained unchanged after HBOC resuscitation in all groups. Following resuscitation, the peak MAP was higher in the High P50 Poly OR-Hb group ( 152 ± 13 mmHg) when compared to either the Low or High P50 large MW, (> 128 kDa) HBOC group (119 ± 15 mmHg or 127 ± 18 respectively, P < 0.05 for both).ConclusionsO-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.RésuméObjectifÉvaluer l’effet de l’affinité pour l’oxygène et du poids moléculaire (PM) des transporteurs d’oxygène à base d’hémoglobine (TOBH) avec o-raffinose sur l’apport d’oxygène cérébral et la tension artérielle moyenne (TAM) après une hémorragie et une réanimation chez des rats.MéthodeDes rats anesthésiés à l’isoflurane (n = 6-7 par groupe) ont subi une hémorragie à 30% et une réanimation avec un volume équivalent de l’un des trois différents TOBH suivants : 1) de l’hémoglobine Poly o-raffinose à P50 élevé (Poly OR-Hb, P50 = 70 mmHg) ; 2) 128 Poly OR-Hb à P50 élevé (PM > 128 kDa, P50 = 70 mmHg) et 3) 128 Poly OR-Hb à faible P50 (PM > 128 kDa, P50 = 1 1 mmHg). La tension en oxygène du tissu cérébral hippocampique, le débit sanguin cérébral régional (DSCr), la TAM, la concentration d’hémoglobine totale et la gazométrie du sang artériel ont été mesurés. L’analyse de données, par double ANOVA et tests de Tukey ultérieur, ont permis de déterminer la valeur significative (P < 0,05, moyenne ± SD).RésultatsLa tension en oxygène du tissu hippocampique s’est accrue dans tous les groupes de TOBH après la réanimation. Le DSCr est resté le même dans tous les groupes après la réanimation avec les TOBH. Après la réanimation, la TAM était plus élevée dans le groupe Poly OR-Hb à P50 élevé (152 ± 13 mmHg) comparé au groupe de TOBH de P50 élevé et de grand PM, (> 128 kDa) (119 ± 15 mmHg ou 127 ± 18 respectivement, P < 0,05 pour les deux).ConclusionLes TOBH polymérisés avec o-raffinose, avec ou sans composants de faible PM, ont maintenu l’apport d’oxygène au tissu cérébral après une hémorragie et une réanimation chez des rats. Les TOBH de PM élevé ont montré une baisse de la TAM qui n’a pas nui à l’apport d’oxygène. Aucun effet significatif de l’affinité pour l’oxygène sur la tension en oxygène du tissu cérébral ou sur le débit sanguin n’a été observé.