HYPOVOLEMIC AND CIRCULATORY SHOCK
BLOCKING INFLAMMATORY CYTOKINE MEDIATED HEPATOXICITY
Myocardial infarction protection by Delt-Dvar relies in part on a mechanism(s) involving interaction between delta opioid receptors and KATP channels: We have also demonstrated that infusions of Delt-Dvar could decrease left ventricular infarct volume and enhance left ventricular function in an isolated rat heart model when infused 24 hrs prior to ischemia or in an intact ischemic pig heart model when infused 40 min prior to ischemia. Moreover, by utilizing a KATP channel blocker, glibenclamide, we were able to demonstrate that KATP channel activation was part of the mechanism involved in this extended myocardial ischemia protection. In these studies, isolated, oxygenated Krebs Henselheit Buffer (KHB) perfused rat hearts from rats weighing 350-400g underwent 20 min global ischemia and 120 min reperfusion, at 37ºC in a modified Langendorff mode. Coronary perfusion pressure was maintained at 70 mmHg by regulating coronary flow. Hearts were paced at 5.5 Hz except during ischemia. Left ventricular developed pressure (DP) and left ventricular end diastolic pressure (LVEDP) were measured with a saline filled balloon. Treated rats (n=6 per group) received 2 mg/kg Delt-Dvar via tail vein infusions 24 hrs prior to cardiectomy. Glibenclamide, was infused I.V. at 1.0 mg/kg, 30 min prior to the 24 hrs Delt-Dvar infusion or 30 min prior to cardiectomy following the 24 hr Delt-Dvar infusion. As seen in Figure 1, 24 hr preinfusion of Delt-Dvar resulted in a significant (more than 50%) decrease in left ventricular infarct size compared to saline infused controls. Glibenclamide pretreatment (1.0 mg/kg); at either 24.5 hr or 0.5 hr prior to cardiectomy, effectively abolished infarct size reduction by Delt-D (20 ± 2% group G1 vs 27 ± 5% control group and 23 ± 5% group G2 vs 27 ± 5% control group p=NS).
Figure 1. Delt-D decreases infarct size. Rats were infused with Delt-D, 2 mg/kg in 1.0 mg phosphate buffered saline. Glibenclamide, 1 mg/kg was infused either at 24.5 hr or 0.5 hr prior to cardiectomy, *p < 0.05.
HYPOVOLEMIC AND CIRCULATORY SHOCK
Prehemorrhage Treatment with Delta Specific Opioid: We have recently demonstrated that Delt-Dvar markedly improved survival of severely hemorrhaged rats when infused without concomitant fluid resuscitation 24 hr prior to hemorrhage. In these studies conscious rats with indwelling catheters were hemorrhaged at 3.18 ml/100g over 20 min representing a 53% fixed volume blood loss. Controls (n=14) were infused with 1.0 ml lactated Ringers (LR) solution and Delt-Dvar treated groups (n=22) were infused with 5.0 mg/kg Delt-Dvar in 1.0 ml LR 24 hr prior to hemorrhage. Rats were continuously monitored for heart rate (HR), mean arterial pressure (MAP) and 4 hr survival. Plasma lactate levels were determined at the beginning of hemorrhage (BOH) and end of hemorrhage (EOH). At 240 min, only one of 14 controls (7.1%) survived, while 16 (72.7%) of 22 test rats survived as shown in Fig. 1. No significant changes in HR were noted. MAP of Delt-Dvar - treated rats was significantly increased (p<0.05) at the BOH (129 ± 2.1 mmHg vs 122 ± 2.1 mmHg) and EOH (67.3 ± 3.4 mmHg vs 53.5 ± 2.9 mmHg) as shown in Fig. 2. Lactate levels of the Delt-Dvar - treated group was significantly lower, 8.5 ± 0.5 mmol/L versus 10.58 ± 0.6 mmol/L for controls (p<0.05) as shown in Fig. 3.
Figure 1. Delt-Dvar increases MAP. Comparison of the average MAP (mmHg) over the course of 4.0 hours. The data represent only survivors at each point in time and are displayed as means with standard error of mean. Differences in MAP between groups were found to be statistically* significant at baseline, end of hemorrhage (EOH), 30 minutes and 60 minutes after hemorrhage (*p<0.05). At the 240-minute time point, only one of the original 14 control rats survived while 16/22 of the opioid treated rats survived.
Figure 2. Delt-Dvar increases survival. Comparison of the percentage of survivors for the Deltorphin-D variant and control groups in 30-minute increments over the 4 hours. Kaplan Meier Survival analysis reveals statistically significant differences in survival distribution between the control and Delt-Dvar groups. The three indices Chi-square test values and p-levels were l6.6, p<0.0000 for the 10g rank method; 13.1, p=0.0003 14.7, p=0.0001 for the Tarone-Ware method. While only one of the control rats survived past 150 minutes, 72.7% of the Deltorphin-D variant treated rats were still alive at the end of the experiment.
Figure 3. Delt-Dvar reduces lactate levels. Comparison of the changes in levels of lactic acid (mmol/L) in the arterial blood sampled at the BOH and at the EOH for the Deltorphin-D variant and Control groups, including standard error of mean. Lactic acid levels for controls and Delt-Dvar group were not significantly different at BOH. The Delt-Dvar treated group had a statistically significant lower lactic acid level (p<0.01) than the control at EOH.
Posthemorrhage Treatment with Delta Specific Opioid: We have recently demonstrated that Deltorphin-E (Delt-E) markedly improved survival of severely hemorrhaged rats when infused immediately after hemorrhage without concomitant fluid resuscitation. In these studies, conscious rats with indwelling catheters were hemorrhaged at a fixed total blood volume of 48%. Test rats (n=24) were infused immediately following hemorrhage in the femoral vein with either 2.85, 4.2, 5.5 or 14 mg/kg of Delt-E, a novel delta2 specific decapeptide aa dissolved in 1.0 ml LR. Controls were infused with 1.0 ml LR. Rats were continuously monitored for HR, MAP and 6 hr survival and plasma lactate levels were determined at BOH and within 10 min after Delt-E infusion. We noted a significant (p<0.01) increase in survival time for the 2.85 mg/kg, 4.2 mg/kg, and 5.5 mg/kg Delt-E treated group versus control group (107 ± 1 min, 232 ± 14 min. and 331 ± 18 min vs 50 ± 8 min, respectively). The 14.0 mg/kg dose of Delt-E was found to be toxic with a survival time of 27 ± 7 min vs control 50 ± 8 min as shown in Fig. 4. The maximum MAP recorded during recovery posthemorrhage was significantly higher for the 2.85 mg/kg (p<0.05), 4.2 mg/kg (p<0.05)), and 5.5 mg/kg (p<0.01) treated group versus the saline control group, 50 ± 17 mmHg, 53 ± 11 mmHg, and 58 ± 7 mmHg vs 35 ± 9 mmHg, respectively as shown in Fig. 5. HR was not significantly different from controls. Mean lactic acid levels at EOH for saline controls increased six-fold to 8.88 ± 0.1 mmol/L and four-fold for the 5.5 mg/kg Delt-E treated group to 6.54 ± 1.54 mmol/L (p>0.01) as shown in Fig. 6.
The aforementioned studies indicate that delta2 specific opioids (Delt-Dvar and Delt-E) are highly effective in improving hemodynamic indices and enhancing survival of severely hemorrhaged rats when infused either 24 hr prior to hemorrhage or immediately posthemorrhage without concomitant fluid resuscitation. Moreover, both Delt-Dvar and Delt-E significantly lowered posthemorrhage plasma lactate levels produced via the glycolytic pathway thereby indicating their role as metabolic inhibitors.
Figure 4. Post-severe hemorrhage Delt-E treatment demonstrates dose-dependent recovery of MAP during recompensatory phase. Only the 5.5 mg Delt-E was able to maintain survival during the 6 hr test period. EOD represents end of hemorrhage.
Figure 5. Delta-E dose-dependent increase in survival time (expressed in minutes) after severe hemorrhage.
Figure 6. Delt-E dose dependent decrease in lactic acid concentration (expressed in millimeter concentration). There was no significant difference in lactic acid level at beginning of hemorrhage (BOH) between the control group and any Delt-E group. However, at end of hemorrhage (EOH), only the 5.5 mg/kg Delt-E had significantly lower lactic acid level.
BLOCKING INFLAMMATORY CYTOKINE MEDIATED HEPATOXICITY
Delt-Dvar is Protective Against Endotoxin Shock: We have also shown that Delt-Dvar is protective against endotoxin shock in a mouse model. Endotoxin initiates a cascade of cellular events including free radical production which, in turn, leads to NF-kB activation and the upregulation of NF-kB dependent cytokine production. This results in the massive release of inflammatory cytokines including tumor necrosis factor alpha (TNFα) and interleukin-1 (IL-1). Our studies have demonstrated that the Delt-Dvar is protective against endotoxin shock. Specifically, BLAB/c mice were pretreated with Delt-Dvar or DADLE (a non-specific delta opioid) then injected with galactosamine and lipopolysaccharide (LPS) to induce endotoxin shock. No control mice survived 15 hr, while 3 out of 15 (20%) DADLE-treated mice survived 65 hr and 11 out of 15 (73%) Delt-Dvar survived 65 hr. Thus, Delt-Dvar reduced LPS induced oxidative stress and provided significant protection against endotoxin shock as seen in Table 1.
Table 3: Opioids Protect Against Endotoxin Shock in BALB/c Mice _______________________________________________________________________________
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LPS (100μg/kg), GAL (700mg/kg), LPS/GAL injection at 6.5 hr.
Deltorphin-Dvar and DADLE (4.0mg/kg) injected at 0,2,4,6 and 7.5 hr (n=15 per group)