Lack of replicative senescence in normal rodent glia.

Replicative senescence is thought to be an intrinsic mechanism for limiting the proliferative life-span of normal somatic cells. We show here that rat Schwann cells can be expanded indefinitely in culture while maintaining checkpoints normally lost during the immortalization process. These findings demonstrate that senescence is not an inevitable consequence of extended proliferation in culture.

Diaspirin crosslinked hemoglobin (DCLHb): more effective than lactated Ringer's solution in restoring central venous oxygen saturation after hemorrhagic shock in rats.

Central venous oxygen saturation (ScvO2) has been shown to reflect tissue oxygen consumption in hemorrhagic shock. The purpose of this study was to test whether the "blood substitute" diaspirin crosslinked hemoglobin (DCLHb, Baxter Healthcare, Round Lake, IL) might be more effective than lactated Ringer's solution (LR) at restoring tissue oxygenation, as measured by ScvO2, when used as a resuscitative fluid following hemorrhage. Conscious male Sprague-Dawley rats (250-350 gm) were bled through a jugular venous catheter to a target central venous base deficit of 15 +/- 2 mmol/L. Animals were immediately resuscitated with either 10% DCLHb (1:1) or LR (3:1), based on shed blood volume, followed by a maintenance infusion of LR until completion of the experiment. Central venous blood was sampled at baseline, prior to resuscitation and every 15 minutes for the first hour following resuscitation. While the baseline and pre-resuscitation ScvO2 values were not significantly different between groups, ScvO2 values were greater (P < or = 0.01) in the DCLHb group at all times following resuscitation. Furthermore, DCLHb restored SvO2 to baseline by 15 minutes after resuscitation, whereas LR resuscitation never restored ScvO2 to baseline. Since venous desaturation is one of the major compensatory mechanisms by which oxygen consumption is maintained under conditions of limited oxygen supply, these data suggest that animals resuscitated with DCLHb had a more rapid restoration of tissue oxygenation than those resuscitated with LR in this model of hemorrhagic shock.

Diaspirin cross-linked hemoglobin is efficacious in gut resuscitation as measured by a GI tract optode.

The objective of this study was to compare the efficacy of diaspirin cross-linked hemoglobin (DCLHb) with that of standard resuscitative fluids in restoring intestinal mucosal oxygenation and villous architecture after hemorrhage. Male rats were bled to a base deficit of 5 +/- 2 nmol/l under propofol anesthesia and monitored for 90 minutes postresuscitation with DCLHb, blood, lactated Ringer's solution, albumin, or nothing (DNR) for mucosal oxygen tension (Pmo2) and physiologic and laboratory parameters. Small intestinal histologic specimens were obtained and scored independently by two investigators blinded to therapy on a scale of 0 (normal) to 4 (worst). All treatments restored Pmo2; only DCLHb did so without exceeding baseline values. For untreated rats (DNR), Pmo2 was not restored. Normal mucosal architecture was maintained only in DCLHb-treated rats. As Pmo2 increased, mucosal score improved. In a rat model of controlled hemorrhage, Pmo2 changes measured by an optode correlated with gut histological abnormalities. By these criteria, DCLHb is superior to crystalloid, colloid, and blood in gut resuscitation.

Subcutaneous oxygen tension: a useful adjunct in assessment of perfusion status.

OBJECTIVES: Using a new fluorescence-quenching optode which, unlike earlier oximeters, neither consumes oxygen nor generates heat, we sought to determine the effects of hemorrhage and resuscitation on subcutaneous PO2. Additionally, we compared the effects of resuscitation with diaspirin crosslinked hemoglobin, an oxygen-carrying solution, on subcutaneous PO2 to that of traditional resuscitative fluids. We also compared mean arterial pressure and central venous oxygen saturation, indirect indices of perfusion, to subcutaneous PO2, a direct index of perfusion. DESIGN: Prospective trial, randomized for selection of treatment regimen. SETTING: Shock-trauma laboratory of a medical university. SUBJECTS: Male Sprague-Dawley rats, weighing 260 to 380 g. INTERVENTIONS: Rats were bled 22 mL/kg and resuscitated, 1 min later, with either 66 mL/kg of lactated Ringer's solution, 22 mL/kg of human serum albumin, 22 mL/kg of blood, or 22 mL/kg of diaspirin crosslinked hemoglobin. A fifth group of animals was not resuscitated after hemorrhage. Subcutaneous PO2 and mean arterial pressure were monitored continuously throughout the experiment, while central venous oxygen saturation was measured intermittently. MEASUREMENTS AND MAIN RESULTS: Subcutaneous PO2 decreased in response to hemorrhage and, although it did increase after resuscitation with each fluid, no treatment was able to restore subcutaneous PO2 to baseline within 2 hrs postresuscitation. Subcutaneous PO2 continued to decrease after hemorrhage in the unresuscitated animals. In contrast, mean arterial pressure was restored to baseline values in only blood- and diaspirin crosslinked hemoglobin-treated animals, although this effect was lost within 30 mins in the blood-treated group. Only blood restored the central venous oxygen saturation to baseline values in the early postresuscitation period. CONCLUSIONS: The fluorescence-quenching optode consistently followed changes in subcutaneous PO2 during hemorrhage and after resuscitation. Diaspirin crosslinked hemoglobin performed as well as blood in restoring peripheral perfusion, as measured by subcutaneous PO2, while both of these fluids were superior to either lactated Ringer's solution or albumin. Both whole blood and diaspirin crosslinked hemoglobin restored mean arterial pressure to baseline, although the effect of the latter was of a longer duration. The pressor effect of the crosslinked hemoglobin did not affect peripheral perfusion, as reflected by the values for subcutaneous PO2. Subcutaneous PO2 is a useful adjunct in assessment of the adequacy of peripheral perfusion and may help redefine targets for resuscitation.

Diaspirin crosslinked hemoglobin (DCLHb) attenuates bacterial translocation in rats.

Intestinal barrier function is compromised following severe hemorrhage which may allow bacterial translocation (BT) to occur and subsequently initiate a systemic response leading to multiple system organ failure (MSOF). This study compared BT following hemorrhage and resuscitation with lactated Ringer's solution (LR) or diaspirin crosslinked hemoglobin solution (DCLHb). Rats (250-350 grams) were hemorrhaged to a base deficit of 15 +/- 2 mmol/L and immediately resuscitated with either 3:1 LR or 1:1 DCLHb based on shed blood volume. Four hours following resuscitation, the mesenteric lymph node complex was harvested, homogenized and plated onto MacConkey and Columbia CNA agar culture media. Facultative anaerobic and obligate aerobic bacteria were identified 48 hours later in 11/22 (50%) LR-treated rats and in 4/21 (19%) DCLHb-treated rats (p < or = 0.05). Following resuscitation, base excess (BE) and central venous oxygen saturation (SvO2) were not only restored to baseline but were significantly greater (p < or = 0.05) in DCLHb-treated rats than in LR-treated rats. In a separate group of rats subjected to the same hemorrhage and resuscitation protocol, mean arterial pressure in DCLHb-treated rats, but not LR-treated rats, was restored to baseline by 15 minutes and remained at or above baseline for up to 4 hrs. Twenty-four hour survival was 50% in LR-treated rats and 77% in DCLHb-treated rats (p > 0.05). These data suggest that DCLHb is superior to LR in restoring tissue oxygen delivery, as judged by BE and SvO2. Furthermore, since DCLHb restores oxygen delivery and attenuates BT, early resuscitation with DCLHb may limit gut ischemia and subsequent gut barrier failure and hence prevent the development of sepsis, MSOF and subsequent death.

The efficacy of diaspirin crosslinked hemoglobin solution resuscitation in a model of uncontrolled hemorrhage.

Controversy exists whether early aggressive fluid therapy in the setting of uncontrolled hemorrhage worsens outcome by increasing blood loss from injured vessels. Since diaspirin crosslinked hemoglobin (DCLHb) is a vasoactive, oxygen-carrying solution, we compared the effects of DCLHb with other resuscitative fluids on blood loss, hemodynamics, and tissue oxygen delivery in a model of uncontrolled hemorrhage. Anesthetized rats (250-350 g) were subjected to a 50% tail transection and resuscitated 15 minutes later with 1:1 DCLHb, 3:1 lactated Ringer's solution (LR), 1:1 hypertonic saline (7.5% HTS), or 1:1 human serum albumin (8.3% HSA) based on initial volume of blood loss (average 4.7 +/- 0.3 mL/kg). An unresuscitated group served as a control. Cumulative blood loss was measured at 5 hours postresuscitation. By 15 minutes after tail transection, mean arterial pressure (MAP) decreased 19.2 +/- 3.8 mm Hg from the baseline value (102 +/- 5 mm Hg). The DCLHb solution restored and maintained MAP and subcutaneous tissue oxygen tension at baseline values better than all other resuscitative fluids. Although blood loss in DCLHb-treated animals was greater than in unresuscitated animals, it was no different from other resuscitative fluids and less than with HSA. There was no difference in 24-hour survival between all treatment groups. In conclusion, DCLHb elevates MAP but does not exacerbate blood loss or compromise tissue oxygen delivery compared with other resuscitative fluids in this model of uncontrolled hemorrhage.

Characterization of the hemodynamic response to intravenous diaspirin crosslinked hemoglobin solution in rats.

Diaspirin crosslinked hemoglobin solution (DCLHB) has potential for clinical use as an oxygen-carrying solution because of its excellent oxygen transport properties and biochemical stability. The present study characterizes the effects of intravenous infusions of 0.625-40 mL/kg (62.5-4000 mg/kg) DCLHb on mean blood pressure (MAP) and heart rate (HR) in conscious rats. DCLHb at all doses tested except 62.5 mg/kg was associated with an immediate increase in MAP (25-30% above baseline) that peaked between 20-30 minutes after infusion and returned to baseline within 120-300 minutes in a dose-dependent manner. Maximum MAP achieved was in the range of 129 +/- 7 to 140 +/- 7 mm Hg and there was no statistically significant difference in the response between doses. HR responded in a reciprocal manner to changes in MAP. Volume- and oncotic-matched infusions of LR and albumin did not alter MAP or HR. Slow infusion (0.34 mL/min) of DCLHb appeared to blunt the magnitude of the pressor response when compared to bolus injection (< 10 sec). DCLHb administration is associated with a pressor response that is not due to volume load, oncotic pressure, or rate of infusion, suggesting that it is intrinsic to the modified hemoglobin molecule and pharmacologic in nature.

Use of base deficit to compare resuscitation with lactated Ringer's solution, Haemaccel, whole blood, and diaspirin cross-linked hemoglobin following hemorrhage in rats.

Base deficit (BD) has been shown to be a sensitive measure of the degree and duration of inadequate perfusion. We developed a rat model of hemorrhagic shock based on achieving a fixed BD of 13 +/- 1 mmol/L before resuscitation. Using this model, we compared the efficacy of resuscitation with lactated Ringer's solution (LR), Haemaccel (a colloid), and whole blood with that of diaspirin cross-linked hemoglobin (DCLHb, Baxter Healthcare Corp.) by evaluating improvements in BD and restoration of base excess (BE, positive correlate of BD) for 60 minutes following resuscitation. The DCLHb was superior to LR and Haemaccel in restoring and maintaining BE following resuscitation, and was able to restore BE as rapidly as whole blood at half the volume. At 60 minutes, DCLHb at twice the shed blood volume maintained BE at higher (more positive) values compared with all other treatment groups. We conclude that DCLHb is at least as effective as whole blood and superior to LR and Haemaccel in restoring BE within the first 60 minutes following resuscitation in this hemorrhagic shock model.

A role for endothelin and nitric oxide in the pressor response to diaspirin cross-linked hemoglobin.

Diaspirin-cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp) has potential for clinical use as a hemoglobin-based oxygen-carrying solution. We have previously shown that DCLHb administration is associated with a self-limiting increase in mean arterial pressure (MAP). Based on in vitro studies with other hemoglobin solutions, this vasopressor effect is thought to be mediated at least in part by the release or inhibition (or both) of endothelium-derived vasoactive substances. The purpose of our studies was to determine the role of endothelin (ET), a potent vasoconstrictor peptide, and nitric oxide, a vasodilator, in mediating the pressor effect of DCLHb in conscious rats. Intravenous administration of DCLHb has been shown to elicit an immediate increase in MAP that peaks within 30 minutes and returns to baseline by 300 minutes in a dose-dependent fashion. Phosphoramidon, an inhibitor of proendothelin conversion to ET, attenuated the elevation of MAP when administered before DCLHb. Administration of cyanomet DCLHb, a DCLHb molecule that is unable to interact with NO, was not associated with an elevation of MAP. L-arginine, the substrate for NO synthesis, and nitroglycerin, an NO donor, significantly reduced MAP when infused 15 minutes after DCLHb administration. Based on these findings, we conclude that the DCLHb-induced elevation of MAP in vivo is mediated at least in part by ET and the inhibition of NO. Although these data support earlier reports of hemoglobin binding NO, this is the first report of the pressor response to hemoglobin being attenuated by an agent that blocks the conversion of proendothelin to ET.

Hypertonic saline resuscitation detrimentally affects renal function and survival in dehydrated rats.

The purpose of our studies was to determine whether hypertonic (7.5%) saline (HTS) resuscitation is effective in the setting of dehydration. We compared the effects of HTS (5 cc/kg) to those of Ringer's lactate (RL; 45 cc/kg) on renal function, following resuscitation from hypovolemia in hydrated (free access to food/water) vs. dehydrated (food/water restricted) rats (300-350 g). Renal failure was produced by hemorrhage (15 cc/kg) plus renal artery occlusion (25 min) followed by fluid resuscitation. Dehydration was confirmed by hemoconcentration and weight loss (8-10%). Renal function was assessed at 24 hr using 14C-inulin clearance (Cin) measurements. In hydrated animals, the Cin of RL-treated rats (625 +/- 54 microliters/min/100 g; n = 12) was no different from the Cin in HTS-treated rats (517 +/- 48 microliters/min/100 g; n = 13). Among dehydrated rats, Cin in HTS-treated rats (n = 6) was significantly lower (P < or = 0.05) than in RL-treated rats (n = 5) (117 +/- 33 microliters/min/100 g vs. 542 +/- 84 microliters/min/100 g, respectively). Cin in dehydrated RL-treated rats was not significantly different from that in hydrated RL-treated rats. Furthermore, in dehydrated animals, nine of nine resuscitated with RL survived, compared to six of 13 resuscitated with HTS. All hydrated animals survived. In summary, renal failure was ameliorated by RL and worsened by HTS resuscitation in dehydrated rats. Furthermore, mortality was increased in dehydrated animals resuscitated with HTS compared to RL.

Cross-linked hemoglobin solution as a resuscitative fluid after hemorrhage in the rat.

Intramolecularly (alpha-alpha) cross-linked hemoglobin has been reported to have oxygen transport properties similar to those of whole blood. The present study evaluated the efficacy of diaspirin alpha-alpha cross-linked hemoglobin solution as a resuscitation fluid, with heart rate, mean arterial pressure, and transcutaneous oxygen tension as the study parameters. Rats were bled and approximately one third of their total blood volume (20 ml/kg) was removed while they were anesthetized; they were then resuscitated with 14% hemoglobin solution. Animals that received either 10 mg/kg (n = 10) or 20 mg/kg (n = 10) of hemoglobin solution responded quickly and positively to the infusions: mean arterial pressure (which had dropped to less than 40% of prehemorrhage levels) returned to baseline within 2 minutes of initiating infusion; by 4 minutes, the mean arterial pressures of the hemoglobin-infused groups were significantly higher (p less than or equal to 0.05) than those in both the autologous shed blood (n = 8) and lactated Ringer's (n = 10) groups. The heart rate and transcutaneous oxygen tension responses in both the half-volume and full-volume replacement hemoglobin groups matched the response to autologous shed blood throughout the hour of observation. The favorable hemodynamic response to infusion of cross-linked hemoglobin solution after hemorrhage suggests that this material is comparable to autologous shed blood and superior to lactated Ringer's solution as a resuscitative fluid as assessed in this model.

Calcium administration increases the mortality of endotoxic shock in rats.

Calcium chloride is administered frequently to critically ill patients to improve cardiac output and BP. However, Ca has been implicated in the pathophysiology of shock and ischemic disorders. To test the hypothesis that Ca may be deleterious to shock outcome, we studied the effects of CaCl and Ca chelator (EGTA) infusions on mean arterial pressure (MAP) responses to endotoxin and 24-h survival in rats. Increasing ionized Ca from 4.1 +/- 0.06 to 4.9 +/- 0.20 and 8.5 +/- 0.52 mg/dl progressively increased endotoxin lethality from 20% to 37% and 80%, respectively. This occurred despite slight improvements in MAP in hypercalcemic rats. Conversely, hypocalcemia (3.6 +/- 0.08 mg/dl) lowered endotoxin-induced mortality to 0 without significant effects on MAP. Ca and EGTA infusions alone were not associated with any mortality. Although Ca administration may improve MAP, it significantly increases mortality associated with endotoxic shock in rats. Based on these observations, we advise caution when using Ca in patients with sepsis.

Naloxone potentiates epinephrine's pressor actions in endotoxemic rats.

Naloxone's pressor effects in shock may be mediated through antagonism of endogenous opioid inhibition of sympathoadrenal catecholaminergic systems. Since circulating catecholamine levels are not further elevated by naloxone in endotoxemic animals, it is possible that naloxone acts upon opiate receptors to enhance catecholamine actions at the receptor or postreceptor level. To investigate this hypothesis, we sought to determine whether naloxone treatment would augment the pressor actions of exogenous catecholamines (epinephrine) in normal and endotoxemic rats. Naloxone (3 mg/kg intravenous [i.v.] bolus followed by 3 mg/ml/hr infusion) significantly augmented the pressor response to 10, 20, and 50 micrograms/kg of i.v. epinephrine by 69%, 48% and 14%, respectively, in endotoxin (5 mg/kg, LD20, i.v.) -treated rats but not in normal rats. Likewise, the duration of the pressor response to epinephrine was significantly increased by naloxone. These findings suggest that the coadministration of naloxone and epinephrine may be of benefit in the treatment of shock.

Intrathecal dynorphin A1-13 and dynorphin A3-13 reduce rat spinal cord blood flow by non-opioid mechanisms.

Radiolabeled microspheres were used to examine the effects of paralytic intrathecal doses of dynorphin A (Dyn A1-13) and Dyn A3-13 on rat brain and spinal cord blood flows and cardiac output. Dyn A1-13 produced significant dose-related reductions in blood flow to lumbosacral and thoracic spinal cord without altering cardiac output and blood flow to brain and cervical spinal cord. Naloxone failed to block these effects. Dyn A3-13, which lacks opioid activity, also significantly reduced blood flow in lumbosacral spinal cord. Thus, the paralytic effects of Dyn A in the rat may involve reductions in spinal cord resulting from non-opioid actions of Dyn A.

Intrathecal dynorphin A (1-13) and (3-13) reduce spinal cord blood flow by non-opioid mechanisms.

Dynorphin A (Dyn A)-related peptides have been implicated in the pathophysiology of spinal cord injury in part because their intrathecal (i.t.) injection causes hindlimb paralysis. The effects of paralytic doses of i.t. Dyn A (1-13) and Dyn A (3-13) on spinal cord blood flow and cardiac output were examined in rats using radiolabeled microspheres. Both Dyn A (1-13) and Dyn A (3-13) significantly reduced blood flow in lumbosacral spinal cord without altering cardiac output. Pretreatment with naloxone failed to block these reductions in blood flow. Thus, the paralytic effects of Dyn A may result from non-opioid actions of Dyn A to reduce spinal cord perfusion.

Central glucagon antagonizes morphine- and stress-induced antinociception in the mouse.

Previously, we have demonstrated that glucagon antagonizes morphine-induced antinociception in the rat (Malcolm et al. 1985). In the present studies we have shown that central injection of glucagon antagonizes morphine- and stress-induced antinociception in the mouse. Since stress-induced antinociception is mediated by the activation of endogenous opioid systems, these findings provide the first evidence for glucagon's ability to antagonize endogenous opioid actions.

Dopaminergic inhibition of pituitary beta-endorphin-like immunoreactivity secretion in the rat.

A pharmacologic approach was used to examine the possible role of dopamine neurons in the regulation of pituitary beta-endorphin-like immunoreactivity (beta-END-LI) secretion in the rat. Blockade of dopamine receptors by haloperidol or pimozide treatment evoked dose- and time-related increases in plasma levels of beta-END-LI. Physical immobilization increased circulating beta-END-LI six-fold and pretreatment with dopamine receptor agonists (bromocriptine or pergolide) significantly attenuated this rise without affecting plasma beta-END-LI levels in non-stressed animals. Dopaminergic drugs and immobilization produced similar effects on circulating PRL as on beta-END-LI. However, the magnitude of change in levels of PRL was generally greater than the change in beta-END-LI. The present findings suggest that dopaminergic neurons inhibit the release of pituitary beta-END-LI as well as PRL in the rat.

Serial hexachlorophene blood levels in the premature infant.

The concentration of hexachlorophene was determined in serial blood samples taken from seven premature infants washed with pHisoHex. Results indicated that after a single wash with 5ml of pHisoHex, blood concentrations reached a maximum of 0.75-1.20mug/ml two to four days after application. The results obtained in this study confirm that the dermal absorption of hexachlorophene is greater in premature than in full-term infants. Use of hexachlorophene for infants. Use of hexachlorophene for infants of birth weight less than 2kg has now been discontinued at Queen Mary Hospital and the amount of pHisoHex used in the initial toileting of infants with birth weight more than 2kg has been limited to a single application of 5ml.