Awake Hamsters Research Articles

The cooling tube: A novel small animal model of systemic hypothermia in awake Syrian Golden Hamsters (mesocricetus auratus).

Hypothermia is increasingly used as a therapeutic strategy in a diversity of clinical scenarios. Its impact on mammalian physiology, particularly on the microcirculatory changes of critical organ systems, are, however, incompletely understood. Close examination of the literature reveals a marked paucity of small animal models of rapid systemic hypothermia. All published models introduce important microvascular confounders by investigating either local cooling processes or using anaesthetised animals. Here we present the first rapid systemic hypothermia model in an awake hamster. We developed a waterstream cooled copper tube system for standardized systemic temperature control. With this novel system core body temperature (Tc) in 14 awake animals could be precisely stabilised at temperatures of 30°C and 18°C (7 animals, respectively) within 10-20 min. Rewarming was achieved over 10-15 min. Tolerance of the procedure was excellent. Hamsters did not show any behavioural changes in the mild hypothermia group. In the deep hypothermia group 6 of 7 animals regained normal behaviour within 2-11 hs. As hypothermia was induced in dorsal skinfold chamber bearing animals this model seems suitable for investigation of microcirculatory purposes.Advantages over previously established experimental hypothermia models are significant. Amongst these, the possibility of visualization of microcirculation, the lack of microcirculation confounding factors such as anaesthetic drugs, the ability for precise Tc control and rapid induction of hypothermia are prominent.

The Suprachiasmatic Nucleus Gets Split: Why Does Cortisol Respond But Not ACTH?

In mammals, circadian timing is dependent on a pacemaker or clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus (1). The clock is reset daily by environmental lighting transmitted to the SCN from the retina via the retinohypothalamic pathway (2). This mechanism guarantees that daily rhythms in behavior and physiology including hypothalamic-pituitary-adrenal (HPA) activity are coordinated with the daily light-dark cycle. The HPA axis is characterized by a prominent circadian rhythm that is essential for providing circulating glucocorticoids to support metabolic, cardiovascular, and neuronal processes at the onset of daily activity periods. Circadian rhythms in adrenal corticosterone are driven by in-phase rhythms in plasma ACTH and in adrenal sensitivity to ACTH (3). Adrenal sensitivity is modulated by splanchnic neural activity (4, 5). Taken together, these findings have led to the hypothesis that rhythms in adrenal secretion of glucocorticoids are regulated by not only pituitary ACTH but also sympathetic adrenal innervation (6, 7). In the present issue of Endocrinology, Lilley et al. (8) provide novel results supporting the existence of pathways from the SCN that could subserve dual regulation of glucocorticoid rhythmicity. In this study, Syrian golden hamsters were induced to split rhythms of locomotor activity by exposure to constant light. As shown previously, the split rhythm is manifested as two bouts of locomotor activity separated by approximately 12 h (9, 10). The episodes of locomotion are thought to result from the activity of two coupled circadian oscillators cycling in antiphase ( 12 h out of phase) corresponding to the left and right SCN nuclei (10). By collecting serial blood samples at 1-h intervals throughout a 24-h period in awake hamsters, the study by Lilley et al. (8) showed a clear difference in cortisol rhythms between split and unsplit animals. Whereas unsplit control hamsters showed a single peak of cortisol that preceded a single period of locomotor activity, the split hamsters showed two peaks of cortisol separated by approximately 12 h that were associated temporally with the split rhythm in locomotion. This unique observation is consistent with the hypothesis that dual SCN oscillators are capable of controlling glucocorticoid secretion. Nonetheless, these data do not answer the major question: are there pathways from the SCN that sustain glucocorticoid rhythmicity by activating both HPA and sympathoadrenal systems? To address this issue, the investigators completed another experiment to examine whether peaks in plasma cortisol are associated temporally with peaks in ACTH. To overcome the technical hurdle of obtaining a sufficient sample volume to measure ACTH without activating a hypovolemia-induced stress response, samples were collected at 3-h intervals, a sampling frequency still sufficient to characterize rhythmicity. Results showed that in unsplit control hamsters, a single peak in plasma ACTH was associated with the peak in plasma cortisol. The unexpected finding, however, was that split hamsters showing two peaks in cortisol showed no peaks in plasma ACTH. This finding supports the hypothesis that an extra-ACTH mechanism is responsible for regulating cortisol secretion in the split hamster. The investigators propose that sympathoadrenal control of glucocorticoid secretion is the extra-ACTH mechanism [Fig. 3 (8)]. Although experimental results are not presented, there is compelling evidence to support this conjecture. The adrenal cortex receives sympathetic in-

Impact of Enzymatic Degradation of the Endothelial Glycocalyx on Vascular Permeability in an Awake Hamster Model

Background. The inside of the endothelium is covered by a glycocalyx layer, and enzymatic degradation of this layer induces vascular leakage ex vivo. We hypothesized that enzymatic degrading of the glycocalyx in an in vivo, whole body model, would induce plasma leakage and affect the microcirculation. Methods. Golden Syrian hamsters were divided into an enzyme (hyaluronidase) and a control group. Mean arterial pressure (MAP), heart rate (HR), hematocrit (Hct), base excess (BE), and plasma volume were obtained before, 45 and 120 min after enzyme/saline treatment. Plasma volume was evaluated by the distribution volume of indocyanine green and the microcirculation by functional capillary density (FCD). The enzymatic effect was determined by measuring plasma levels of hyaluronan (HA). Results. There were no differences in MAP, HR, Hct, and BE between the two groups. Enzyme treatment did not induce changes in plasma volume but reduced FCD. There was a 50–100-fold increase in plasma HA, but no relationship was found between HA levels and plasma volume or FCD. Conclusion. Vascular leakage was not confirmed in an in vivo, whole body model after degradation of the endothelial glycocalyx. The microcirculation was affected, but no relationship between plasma levels of HA and FCD was seen.

Hypothyroidism attenuates SCH 23390-mediated depression of breathing and decreases d1 receptor expression in carotid bodies, PVN and striatum of hamsters

Hypothyroidism can lead to depressed breathing. We determined if propylthiouracil (PTU)-induced hypothyroidismin hamsters (HH) altered dopamine D1 receptor expression, D1 receptor-modulated ventilation, and ventilatory chemoreflex activation by hypoxia or hypercapnia. Hypothyroidism was induced by administering 0.04% PTU in drinking water for 3 months. Ventilation was evaluated following saline or 0.25 mg/kg SCH 23390,a D1 receptor antagonist, while awake hamsters breathed normoxic (21% O 2 in N 2), hypoxic (10% O 2in N 2) and hypercapnic (5% CO 2 in O 2)air. Relative to euthyroid hamsters (EH), HH exhibited decreased D1 receptor protein levels in carotid bodies, striatum, and hypothalamic paraventricular nucleus, but not in the nucleus tractus solitarius. Relative to EH, HH exhibited lower ventilation during exposure to normoxia, hypoxia, or hypercapnia, but comparable ventilatory responsiveness to chemoreflex activation. SCH23390 decreased ventilation of EH hamsters exposed to normoxia, hypoxia, and hypercapnia. In HH SCH23390 increased ventilation during baseline normoxia and did not affect ventilation during exposure to hypoxia and hypercapnia, resulting in reduced ventilatory responsivess to chemoreflex activation by hypoxia and hypercapnia. Furthermore, in HH D1 receptor protein levels are decreased in several brain regions and within the carotid bodies. Moreover, D1 receptor-modulation of breathing at rest and during gas exposures were depressed in EH but not HH.

The variability of blood pressure due to small changes of hematocrit

The hematocrit (Hct) of awake hamsters was lowered to 90% of baseline by isovolemic hemodilution using hamster plasma to determine the acute effect of small changes in Hct and blood viscosity on systemic hemodynamics. Mean arterial blood pressure increased, reaching a maximum of about 10% above baseline (8.6 +/- 5.5 mmHg) when Hct decreased 8.4 +/- 1.9% (P < 0.005). Cardiac output increased continuously with hemodilution. These conditions were reached at approximately 60 min after exchange transfusion and remained stationary for 1 h. Peripheral vascular resistance was approximately constant up to a decrease of Hct of about 10% and then fell continuously with lowering Hct. Vascular hindrance or vascular resistance independent of blood viscosity increased by about 20% and remained at this level up to an Hct decrease of 20%, indicating that the vasculature constricted with the lowered Hct. The results for the initial 2-h period are opposite but continuous with previous findings with small increases in Hct. In conclusion, limited acute anemic conditions increase mean arterial blood pressure during the initial period of 2 h, an effect that is quantitatively similar but opposite to the acute increase of Hct during the same period.

A sulfaphenazole-sensitive EDHF opposes platelet-endothelium interactions in vitro and in the hamster microcirculation in vivo

A CYP2C9-dependent endothelium-derived hyperpolarizing factor (EDHF) controls blood flow in many microvascular beds of various species by targeting vascular smooth muscle potassium channels. Since platelets express the same channels, we tested whether EDHF hyperpolarizes platelets and exerts an antithrombotic function in vivo. Interaction of injected human platelets with the arteriolar wall (platelet-vessel wall interaction, PVWI) was assessed by intravital microscopy in skin muscle of awake hamsters. To understand the mechanisms of EDHF-induced platelet inhibition, we studied whether cultured human umbilical vein endothelial cells overexpressing CYP2C9-mRNA in vitro released a factor that could hyperpolarize human platelets. Under control conditions, there was no firm adhesion of platelets to the arteriolar wall, but temporary PVWI occurred. Local superfusion of the CYP2C9 inhibitor sulfaphenazole, at doses known to block EDHF-dependent dilations, significantly augmented PVWI, as did inhibition of NO synthase. Inhibition of both factors exerted additive effects on PVWI. Likewise, firm adhesion of a small fraction of platelets was observed. The prothrombotic effects of CYP2C9 inhibition in vivo were reversed by exogenous superfusion with 11,12-epoxyeicosatrienoic acids. Hyperpolarization reduced platelet adhesion to endothelial cells under static conditions in vitro and was dependent on calcium-activated potassium channels. The factor also reduced ADP-induced expression of platelet P-selectin, indicating reduction of platelet activity. The arteriolar endothelium in vivo continuously releases a CYP2C9-derived EDHF. This EDHF exerts its effects by hyperpolarization of platelets through activation of K(Ca) channels and reduction of platelet adhesion molecule expression, indicating that hyperpolarization reduces platelet activation. This demonstrates that EDHF is part of the antithrombotic properties of healthy endothelium in vivo.

Diagnostic Frequency Continuous Ultrasonography Directly Mitigates Venular Ischemia Reperfusion Damage

Our objective was to determine the effects of ischemia reperfusion (IR) treatment with continuous-mode diagnostic frequency ultrasonography exposure by assessing leukocyte-endothelial cell interactions, in terms of frequency and relative proportions of rolling and firmly attached leukocytes. Studies were carried out in the awake hamster chamber window preparation. Tourniquet ischemia was implemented by compressing a circular ring on the chamber window tissue for 4 hours, followed by reperfusion. Animals were randomly assigned into one of four groups as follows: IR; IR + ultrasonography; IR + N(omega)-nitro-L-arginine methyl ester (L-NAME); and IR + L-NAME + ultrasonography. Venules were exposed to epi-illumination, videorecorded, and leukocytes were categorized as "rolling"; flowing with endothelial contact; or "immobilized" cells, and counted during digital video-playback in 100-microm length segments. Leukocyte interaction with venular endothelium substantially decreased, during longterm reperfusion (p < 0.05) with ultrasonography exposure. Nitric oxide production inhibition, after L-NAME treatment, and ultrasonography exposure resulted in additional earlier substantially decreased leukocyte-endothelial cell interactions (p < 0.05). Venular function improvement, after IR damage, is a primary benefit derived from continuous-mode diagnostic frequency ultrasonography exposure. Although decreased interaction of adherent leukocytes can also be attributed to enhanced arteriolar flow, reduced interaction of rolling leukocytes is an immediate consequence of ultrasonography exposure.

Lowering of Blood Pressure by Increasing Hematocrit with Non–Nitric Oxide–Scavenging Red Blood Cells

Isovolemic exchange transfusion of 40% of the blood volume in awake hamsters was used to replace native red blood cells (RBCs) with RBCs whose hemoglobin (Hb) was oxidized to methemoglobin (MetHb), MetRBCs. The exchange maintained constant blood volume and produced different final hematocrits (Hcts), varying from 48 to 62% Hct. Mean arterial pressure (MAP) did not change after exchange transfusion, in which 40% of the native RBCs were replaced with MetRBCs, without increasing Hct. Increasing Hct with MetRBCs lowered MAP by 12 mm Hg when Hct was increased 12% above baseline. Further increases of Hct with MetRBCs progressively returned MAP to baseline, which occurred at 62% Hct, a 30% increase in Hct from baseline. These observations show a parabolic "U" shaped distribution of MAP against the change in Hct. Cardiac index, cardiac output divided by body weight, increased between 2 and 17% above baseline for the range of Hcts tested. Peripheral vascular resistance (VR) was decreased 18% from baseline when Hct was increased 12% from baseline. VR and MAP were above baseline for increases in Hct higher than 30%. However, vascular hindrance, VR normalized by blood viscosity (which reflects the contribution of vascular geometry), was lower than baseline for all the increases in Hct tested with MetRBC, indicating prevalence of vasodilation. These suggest that acute increases in Hct with MetRBCs increase endothelium shear stress and stimulate the production of vasoactive factors (e.g., nitric oxide [NO]). When MetRBCs were compared with functional RBCs, vasodilation was augmented for MetRBCs probably due to the lower NO scavenging of MetHb. Consequently, MetRBCs increased the viscosity related hypotension range compared with functional RBCs as NO shear stress vasodilation mediated responses are greater.

New phosphorescence quenching oxygen measurements technique yields unusual tissue and plasma Po2 distributions

To the Editor: Wilson et al. (9) report using a novel group of chromophores for measuring oxygen pressure in the interstitial space and blood plasma, proposing that the histogram of PO2 volume distribution measured in the thigh muscle of mice can be used to 1) describe the partition of tissue and intravascular PO2 and 2) characterize the difference between intra- and extravascular PO2 or the vessel wall gradient. Both contentions would appear to be at odds with the results and data presented. Their histograms (Fig. 2) show intra- and extravascular PO2 volume fractions of 10 and 20%, respectively, with PO2 values of 100 –140 Torr. The maximum PO2 in arterial blood leaving the lungs is 100 mmHg, and the existence of a longitudinal gradient downstream is a well established feature of PO2 in the vasculature (8). Even in small animals, it is not clear how the PO2 of blood in the vasculature or the tissue of the limbs can exceed PO2 of blood in the lungs. A measurement technique that records such high PO2 values in the interstitial and the intravascular space raises questions about how the measurements relate to the distribution of PO2 in the tissue reported by previous investigators. The review of Tsai et al. (8) on oxygen gradients in the microcirculation shows that the highest PO2 value recorded in 12 studies of arterioles up to 100 m using either the micro

Refinement of cardiac NMR imaging in awake hamsters: proof of feasibility and characterization of cardiomyopathy

The goal of this study was to demonstrate the feasibility of cardiac NMR imaging in conscious hamsters and its usefulness in evaluating cardiac abnormalities in a small-animal model of cardiomyopathy. Awake hamsters, controls and cardiomyopathic ones (CHF 147), were immobilized in a dedicated holder. Half-Fourier single-shot FSE imaging, with outer-volume suppression and 'black-blood' contrast provided images free from motion artifact with good visualization of cardiac anatomy at any point in the cardiac cycle. Series of double-oblique views were acquired with or without electrocardiograph gating. Image acquisition time was 55 ms, with an in-plane resolution of 470 x 625 microm2. Left ventricular volumes, ejection fraction, and myocardium NMR signal heterogeneity were compared in CHF 147 and control hearts. Left ventricles of CHF 147 hamsters were dilated, as indicated by the increase in end-diastolic cavity volume (299 +/- 79 mm3 compared with the controls (141 +/- 39 mm3; P = 0.0002). Left ventricular ejection fraction was largely reduced (45 +/- 9% vs 86 +/- 4%; P < 0.0001). The NMR signal distribution at an effective echo time of 41 ms was more heterogeneous in the myocardial wall of CHF 147 hamsters than in controls (1.87 +/- 0.37 a.u. vs 0.98 +/- 0.12 a.u., respectively; P = 0.0002). This study is a refinement of animal experimentation, as it demonstrates for the first time that characteristic features of cardiac pathology can be evaluated with ultra-fast NMR imaging in conscious small rodents.

EARLY RESUSCITATION WITH POLYMERIZED BOVINE HEMOGLOBIN REVERSES ACIDOSIS, BUT NOT PERIPHERAL TISSUE OXYGENATION, IN A SEVERE HAMSTER SHOCK MODEL

Awake hamsters equipped with the dorsal window chamber preparation were subjected to hemorrhage of 50% of the estimated blood volume. Initial resuscitation (25% of estimated blood volume) with polymerized bovine hemoglobin (PBH) or 10% hydroxyethyl starch (HES) occurred in concert with an equivolumetric bleeding to simulate the early, prehospital setting (exchange transfusion). Resuscitation (25% of estimated blood volume) without bleeding was performed with PBH, HES, or autologous red blood cells (HES-RBCs). Peripheral microcirculation, tissue oxygenation, and systemic hemodynamic and blood gas parameters were assessed. After exchange transfusion, base deficit was -8.6 +/- 3.7 mmol/L (PBH) and -5.1 +/- 5.3 mmol/L (HES) (not significant). Functional capillary density was 17% +/- 6% of baseline (PBH) and 31% +/- 11% (HES) (P < 0.05) and arteriolar diameter 73% +/- 3% of baseline (PBH) and 90% + 5% (HES) (P < 0.01). At the end, hemoglobin levels were 3.7 +/- 0.3 g/dL with HES, 8.2 +/- 0.6 g/dL with PBH, and 10.4 +/- 0.8 g/dL with HES-RBCs (P < 0.01 HES vs. PBH and HES-RBCs, P < 0.05 PBH vs. HES-RBCs). Base excess was restored to baseline with PBH and HES-RBCs, but not with HES (P < 0.05). Functional capillary density was 46% +/- 5% of baseline (PBH), 62% + 20% (HES-RBCs), and 36% +/- 19% (HES) (P < 0.01 HES-RBCs vs. HES). Peripheral oxygen delivery and consumption was highest with HES-RBCs, followed by PBH (P < 0.05 HES-RBCs vs. PBH, P < 0.01 HES-RBCs and PBH vs. HES). In conclusion, the PBH led to a correction of base deficit comparable to blood transfusion. However, oxygenation of the peripheral tissue was inferior with PBH. This was attributed to its negative impact on the peripheral microcirculation caused by arteriolar vasoconstriction.

Increased cardiac output and microvascular blood flow during mild hemoconcentration in hamster window model

The effect of small hematocrit (Hct) increases on cardiac index (cardiac output/body wt) and oxygen release to the microcirculation was investigated in the awake hamster window chamber model by means of exchange transfusions of homologous packed red blood cells. Increasing Hct between 8 and 13% from baseline increased cardiac index by 5-31% from baseline (P < 0.05) and significantly lowered systemic blood pressure (P < 0.05). The relationship between Hct and cardiac index is described by a second-order polynomial (R2 = 0.84; P < 0.05) showing that Hct increases up to 20% from baseline increase cardiac index, whereas increases over 20% from baseline decrease cardiac index. Combining this data with measurements of blood pressure allowed to determine total peripheral vascular resistance, which was a minimum at 8-13% Hct increase and was described by a second-order polynomial (R2 = 0.83; P < 0.05). Oxygen measurements in arterioles, venules, and the tissue at 8-13% Hct increase were identical to control; thus, as a consequence of increased flow and oxygen-carrying capacity, oxygen delivery and extraction increased, but the change was not statistically significant. Previous results with the same model showed that the observed effects are related to shear stress-mediated release of nitric oxide, an effect that should be also present in the heart microcirculation, leading to increased blood flow, myocardial oxygen consumption, and contractility. We conclude that a minimum viscosity level is necessary for generating the shear stress required for maintaining normal cardiovascular function.

Nitric oxide regulation of microvascular oxygen exchange during hypoxia and hyperoxia

The objective of this work was to test the hypothesis that the limitation of nitric oxide (NO) availability accentuates microvascular reactivity to oxygen. The awake hamster chamber window model was rendered hypoxic and hyperoxic by ventilation with 10 and 100% oxygen. Systemic and microvascular parameters were determined in the two conditions and compared with normoxia in a group receiving the NO scavenger nitronyl nitroxide and a control group receiving only the vehicle (saline). Mean arterial blood pressure did not change with different gas mixtures during infusion of the vehicle, but it increased significantly in the NO-depleted group. NO scavenging increased the reactivity of microvessels to the changed oxygen supply, causing the arteriolar wall to significantly increase oxygen consumption. Tissue Po2 was correspondingly significantly reduced during NO scavenger infusion. The present findings support the hypothesis that microvascular oxygen consumption is proportional to oxygen-induced vasoconstriction. The effect of oxygen on vascular tone is modulated by NO. As a consequence, NO acts as a regulator of the vessel wall oxygen consumption. The vessel wall consumes oxygen in proportion to the local Po2, and an impairment of NO availability renders the circulation more sensitive to changes in the oxygen supply.

In Vivo Phosphorescence Imaging of pO2 Using Planar Oxygen Sensors

Oxygen-dependent quenching of luminescence of metal porphyrin complexes has been used to image the pO(2) distribution over tumor and normal tissue. An experimental setup is described using a platinum(II)-octaethyl-porphyrin immobilized in a polystyrene matrix as transparent planar sensor. Sensitivity over a broad range is high at low pO(2) values (+/- 0.2 mm Hg at 0 mm Hg; +/- 1.5 mm Hg at 160 mm Hg pO(2)). Due to intrinsically referencing via lifetime encoding there was no modification of the sensor response in vivo in the dorsal skinfold chamber model with amelanotic melanoma (A-MEL-3) in awake hamsters when compared to the in vitro calibration. pO(2) measurements over normal tissue (25.8 +/- 5.1 mm Hg) and tumor tissue (9.2 +/- 5.1 mm Hg) were in excellent agreement with previous results obtained in this model using a surface multiwire electrode. Using the presented method the surface pO(2) distribution can be mapped with a high temporal resolution of approximately 100 ms and a spatial resolution of at least 25 mu m. Moreover, the transparent sensor allows the simultaneous visualization of the underlying microvasculature.

Paradoxical hypotension following increased hematocrit and blood viscosity

Hematocrit (Hct) of awake hamsters and CD-1 mice was acutely increased by isovolemic exchange transfusion of packed red blood cells (RBCs) to assess the relation between Hct and blood pressure. Increasing Hct 7-13% of baseline decreased mean arterial blood pressure (MAP) by 13 mmHg. Increasing Hct above 19% reversed this trend and caused MAP to rise above baseline. This relationship is described by a parabolic function (R2 = 0.57 and P < 0.05). Hamsters pretreated with the nitric oxide (NO) synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) and endothelial NOS-deficient mice showed no change in MAP when Hct was increased by <19%. Nitrate/nitrite plasma levels of Hct-augmented hamsters increased relative to control and L-NAME treated animals. The blood pressure effect was stable 2 h after exchange transfusion. These findings suggest that increasing Hct increases blood viscosity, shear stress, and NO production, leading to vasodilation and mild hypotension. This was corroborated by measuring A1 arteriolar diameters (55.0 +/- 21.5 microm) and blood flow in the hamster window chamber preparation, which showed statistically significant increased vessel diameter (1.04 +/- 0.1 relative to baseline) and microcirculatory blood flow (1.39 +/- 0.68 relative to baseline) after exchange transfusion with packed RBCs. Larger increases of Hct (>19% of baseline) led blood viscosity to increase >50%, overwhelming the NO effect through a significant viscosity-dependent increase in vascular resistance, causing MAP to rise above baseline values.

Microvascular oxygen delivery and consumption following treatment with verapamil

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle relaxation to oxygen consumption of the microvascular wall during verapamil-induced vasodilatation. Verapamil HCl delivered in a 0.1 mg/kg bolus injection followed by a continuous infusion of 0.01 mg.kg(-1).min(-1) caused significant arteriolar dilatation, increased microvascular flow and functional capillary density, and decreased arteriolar vessel wall transmural Po(2) difference. Verapamil caused tissue Po(2) to increase from 25.5 +/- 4.1 mmHg under control condition to 32.0 +/- 3.7 mmHg during verapamil treatment. Total oxygen released by the microcirculation to the tissue remained the same as at baseline. Maintenance of the same level of oxygen release to the tissue, increased tissue Po(2), and decreased wall oxygen concentration gradient are compatible if vasodilatation significantly lowers vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. These findings show that treatment with verapamil, which increases oxygen supply through vasodilatation, may further improve tissue oxygenation by lowering oxygen consumption of the microcirculation.

Hyperosmotic-hyperoncotic versus hyperosmotic-hyperviscous: small volume resuscitation in hemorrhagic shock.

The aim of this study was to test the effects of using a high-viscosity fluid after small-volume hyperosmotic resuscitation from hemorrhagic shock and to compare this to hyperosmotic followed by hyperoncotic resuscitation. Studies were made in the awake hamster window chamber preparation with the animals subjected to hemorrhage of 50% of blood volume and resuscitated with a small volume of a 7.5% NaCl solution, which was followed within minutes by infusion of 25% of withdrawn volume of either 0.7% or 0.8% alginate solutions (A0.7%, 7.6 cp; and A0.8%, 10.2 cp) or 5% hydroxyethyl starch (HES 5%, 2.1 cp). All modalities of resuscitation returned blood pressure to near baseline values in 5 min, which remained elevated after 90 min with A0.7% and A0.8% but returned to near shock values in 15 min with HES 5%. Microvascular flow and functional capillary density (FCD) followed the same pattern, being significantly higher for the alginate solutions than HES 5% after 90 min. Plasma viscosity 90 min after resuscitation was 2.1 and 2.6 cp for A0.7% and A0.8%, respectively, and 1.1 cP for HES 5%. There was an apparent directly proportional relationship between the concentration of alginate and blood pressure recovery, with blood pressure near normal with A0.8%, and approximately 20 mmHg lower with A0.7%. The recovery of microvascular flow and FCD, although showing a trend toward being more effective with A0.8%, was not significantly different from A0.7% but statistically different and improved relative to HES 5%. The high-viscosity fluids provide a novel small-volume method of resuscitation that maximizes microvascular perfusion for extended periods until surgical control of bleeding is possible. Results show that high-plasma-viscosity resuscitation provides a more consistent and prolonged resuscitation than hyperoncotic treatment. The increase in viscosity presents a gradual recovery in blood pressure and may be used as an alternative for small-volume hypotensive resuscitation, increasing tissue perfusion while potentially limiting hemorrhage in vascular injuries of the major blood vessels.

Early postdenervation depolarization develops faster at endplates of hibernating golden hamsters where spontaneous quantal and non-quantal acetylcholine release is very small

The hyperpolarization produced by the application of curare to the postsynaptic membrane of the diaphragm neuromuscular synapse (H-effect) is a measure of non-quantal release (NQR) of acetylcholine (ACh) from the motor nerve ending. In mouse diaphragm, H-effect was 9.3 mV, significantly lower in awake hamsters (7.1 mV) and very small (1.1 mV) in hibernating hamsters. Also, the initial resting membrane potential (RMP) after dissection was highest in mouse (81.5 mV, inside negative), significantly smaller in awake hamsters (77.9 mV) and lowest in hibernating hamsters (75.1 mV). The early postdenervation depolarization of muscle fiber RMP to about 66-68 mV developed with half-decay time (T1/2) of 120 min in mouse, more rapidly in active hamsters (T1/2=60 min) and even faster in hibernating hamsters (T1/2=25 min) muscles. This reciprocal correlation between the H-effect and the rate of early depolarization indicates that non-quantal release is important for maintaining the resting membrane potential [Vyskocil, F. 2003. Early postdenervation depolarization is controlled by acetylcholine and glutamate via nitric oxide regulation of the chloride transporter. Neurochem. Res. 28, 575-585]. The amplitude of H-effect in mouse and hamster was proportional to the spontaneous quantal release. The frequency of miniature endplate potentials was highest in mouse (1.6 s-1), much smaller in awake hamsters (0.51 s-1) and very small in hibernating hamsters (0.08 s-1). This is in accordance with the idea that non-quantal release depends on the number of vesicles fused with the presynaptic membrane during quantal release [Edwards et al., 1985; Ferguson, S.M., Savchenko, V., Apparsundaram, S., Zwick, M., Wright J., Heilman, C.J., Yi, H., Levey, A.I., Blakely R.D. Vesicular localization and activity-dependent trafficking of presynaptic choline transporters. J. Neurosci. 23 (2003) 9697-9709].

Increased tissue PO2 and decreased O2 delivery and consumption after 80% exchange transfusion with polymerized hemoglobin.

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2 after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2 delivery, O2 extraction, and tissue Po2 at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue Po2 was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2 release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2 metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.

Oxygen distribution in microcirculation after arginine vasopressin-induced arteriolar vasoconstriction.

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle contraction to oxygen consumption of the microvascular wall during arginine vasopressin (AVP)-induced vasoconstriction. AVP was infused intravenously at the clinical dosage (0.0001 IU.kg(-1).min(-1)) and caused a significant arteriolar constriction, decreased microvascular flow and functional capillary density, and a substantial rise in arteriolar vessel wall transmural Po(2) difference. AVP caused tissue Po(2) to be significantly lowered from 25.4 +/- 7.4 to 7.2 +/- 5.8 mmHg; however, total oxygen extraction by the microcirculation increased by 25%. The increased extraction, lowered tissue Po(2), and increased wall oxygen concentration gradient are compatible with the hypothesis that vasoconstriction significantly increases vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. This conclusion was supported by the finding that the small percentage of the vessels that dilated in these experiments had a vessel wall oxygen gradient that was smaller than control and which was not determined by changes in tissue Po(2). These findings show that AVP administration, which reduces oxygen supply by vasoconstriction, may further impair tissue oxygenation by the additional oxygen consumption of the microcirculation.