Patrick D. Harris

SELECTIVE MICROVASCULAR ENDOTHELIAL CELL DYSFUNCTION IN THE SMALL INTESTINE FOLLOWING RESUSCITATED HEMORRHAGIC SHOCK

Following resuscitation (RES) from hemorrhagic shock (HEM), intestinal microvessels develop progressive vasoconstriction that impairs mucosal blood flow, despite central hemodynamic RES. These events might have clinical consequences secondary to occult intestinal ischemia. We hypothesized that the microvascular impairments were due to progressive endothelial cell dysfunction and an associated reduction in the dilator, nitric oxide (NO), following HEM/RES. Male Sprague-Dawley rats, were monitored for central hemodynamics and the terminal ileum was studied with in vivo videomicroscopy. HEM was 50% of baseline mean arterial pressure (MAP) for 60 min, and RES was with shed blood + 1 volume of normal saline (NS), Following HEM/RES, acetylcholine (10-7, 10-5 M) was topically applied and ileal inflow (A1) and premucosal arteriolar diameters were measured to assess endothelial-cell function at 60 and 120 min post-RES. Normalization of MAP, cardiac output, and heart rate demonstrated adequate systemic resuscitation. Post-RES vasoconstriction developed in A1 (-25%) and premucosal (-28%) arterioles with an associated reduction in A1 flow (-47%). However, there was a selective impairment of endothelialdependent dilation that was manifested only in the smaller premucosal arterioles and not in the inflow, A1 arterioles. This suggests that multiple mechanisms are involved in the development of the post-RES vasoconstriction. The premucosal response was likely mediated by endothelial cell dysfunction, while the A1 response was probably the result of enhanced vasoconstrictor forces. This early microvascular dysfunction might contribute to the late sequelae of intestinal ischemia and might alter microvascular responses to subsequent systemic insults.

Resuscitation regimens for hemorrhagic shock must contain blood.

Endothelial cell dysfunction occurs during hemorrhagic shock (HS) and persists despite adequate resuscitation (RES) that restores and maintains hemodynamics. We hypothesize that RES from HS with crystalloid solutions alone aggravate the endothelial cell dysfunction. To test this hypothesis, anesthetized nonheparinized rats were monitored for hemodynamics, and the terminal ileum was studied with intravital video microscopy. HS was 50% of mean arterial pressure (MAP) for 60 min. Four hemorrhaged groups (10 animals in each group) were randomized for RES: group I with shed blood returned + equal volume of normal saline (NS); group II with shed blood returned + 2× NS; group III with 2× NS only; and group IV with 4× NS only. Two hours post-RES, endothelial cell function was assessed with the endothelial-dependent agonist acetylcholine (ACh, 10−9–10−4 M). Maximum arteriolar diameter was elicited by the endothelial-independent agonist sodium nitroprusside (NTP, 10−4 M). HS caused a selective vasoconstriction associated with low blood flow in inflow A1 arterioles in all hemorrhaged groups. Post-RES vasoconstriction developed in A1 and premucosal arterioles (pA3 and dA3) in all hemorrhaged groups regardless of the RES regimen. However, A1 vasoconstriction and flow were significantly worst in the animals RES with NS alone (−43% and −75%, respectively) compared with those resuscitated with blood and NS (−27% and −57%). Impaired dilation response to ACh was noted in all hemorrhaged animals. However, a significant shift to the right of the dose-response curve (decreased sensitivity) was observed in the animals resuscitated with NS alone irrespective of the RES volume. These animals required at least two orders of magnitude greater ACh concentration to produce a 20% dilation response. For all vessel types, Group II had the best preservation of endothelial cell function. In conclusion, HS causes a selective vasoconstriction of A1 arterioles, which was not observed in A3 vessels. RES from HS results in progressive vasoconstriction in all intestinal arterioles irrespective of the RES regimen. Crystalloid RES after HS does not restore hemodynamics to baseline and is associated with a marked endothelial cell dysfunction. Blood-containing RES regimens preserve and maintain hemodynamics and are associated with the least microvascular dysfunction. Therefore, regimens for RES from HS must contain blood. Endothelial cell dysfunction is not the sole etiologic factor of post-RES microvascular impairment.

Intraperitoneal Resuscitation Improves Intestinal Blood Flow Following Hemorrhagic Shock

ObjectiveTo study the effects of peritoneal resuscitation from hemorrhagic shock. Summary Background DataMethods for conventional resuscitation (CR) from hemorrhagic shock (HS) often fail to restore adequate intestinal blood flow, and intestinal ischemia has been implicated in the activation of the inflammatory response. There is clinical evidence that intestinal hypoperfusion is a major factor in progressive organ failure following HS. This study presents a novel technique of peritoneal resuscitation (PR) that improves visceral perfusion. MethodsMale Sprague-Dawley rats were bled to 50% of baseline mean arterial pressure (MAP) and resuscitated with shed blood plus 2 equal volumes of saline (CR). Groups were 1) sham, 2) HS + CR, and 3) HS + CR + PR with a hyperosmolar dextrose-based solution (Delflex 2.5%). Groups 1 and 2 had normal saline PR. In vivo videomicroscopy and Doppler velocimetry were used to assess terminal ileal microvascular blood flow. Endothelial cell function was assessed by the endothelium-dependent vasodilator acetylcholine. ResultsDespite restored heart rate and MAP to baseline values, CR animals developed a progressive intestinal vasoconstriction and tissue hypoperfusion compared to baseline flow. PR induced an immediate and sustained vasodilation compared to baseline and a marked increase in average intestinal blood flow during the entire 2-hour post-resuscitation period. Endothelial-dependent dilator function was preserved with PR. ConclusionsDespite the restoration of MAP with blood and saline infusions, progressive vasoconstriction and compromised intestinal blood flow occurs following HS/CR. Hyperosmolar PR during CR maintains intestinal blood flow and endothelial function. This is thought to be a direct effect of hyperosmolar solutions on the visceral microvessels. The addition of PR to a CR protocol prevents the splanchnic ischemia that initiates systemic inflammation.

Sepsis alters vessel contraction by adrenoceptor-induced nitric oxide and prostanoid

BACKGROUND Alpha-adrenergic agents contract vascular smooth muscle (VSM) and stimulate endothelial release of secondary factors which modulate VSM contraction. Our study examined constrictor prostanoid (cPN) and nitric oxide (NO) as secondary factors which could alter alpha-1 adrenoceptor-mediated contraction during sepsis. METHODS Sepsis was induced in rats by inoculation of an implanted sponge with Escherichia coli and Bacteroides fragilis. Aortic rings at 24 h from septic (n = 21) and control (n = 21) rats were suspended in physiological salt solution (PSS) with or without blockers to NO (N(G)-monomethylarginine), cPN (mefenamic acid, MFA), or thromboxane A2 (SQ29548). Contraction dose-response curves were generated to determine maximal contraction force (F(max)) and pD2 (sensitivity) to phenylephrine in each experimental group. RESULTS Sepsis increased F(max) to phenylephrine (PHE) (1.18 vs 0.90 g, SEM 0.0703). COX inhibition reduced the F(max) in control (0.63 vs 0.90 g, SEM 0.0675) but not in septic animals (1.19 vs 1.18 g, SEM 0.0433). TXA2 receptor inhibition did not alter F(max) in control (1.017 vs 0.973 g, SEM 0.0959) or septic animals (1.28 vs 1.12 g, SEM 0.0823). NOS inhibition enhanced the F(max) in both nonseptic (2.03 vs 0.83 g, SEM 0.0523) and septic rats (1.96 vs 1.15 g, SEM 0.0526), but did less so in the septic animals. CONCLUSIONS PHE-induced F(max) is determined by a balance between PHE-stimulated VSM alpha-adrenoceptor activity, and PHE-stimulated endothelial release of cPN and NO. Sepsis enhances total PHE-induced F(max) by increasing VSM alpha-adrenoceptor activity and reducing PHE-stimulated endothelial release of dilator NO. Sepsis abolishes the PHE-stimulated endothelial release of cPN. PHE-stimulated cPN is not thromboxane A2, but could be a nonprostanoid dilator in the lipoxygenase (HETE) or cytochrome P450 (EET) pathways.

Role of muscle microvasculature during hyperdynamic and hypodynamic phases of endotoxin shock in decerebrate rats

Microcirculatory derangements in skeletal muscle could act to change cardiac output during endotoxemia. To explore this idea, we measured arteriole and venule responses to low-dose and high-dose endotoxemia in the rat cremaster muscle by direct in vivo videomicroscopy. Our data indicate that cardiac output increased in the low-dose group and decreased in the high-dose group. In both animal groups, a differential arteriolar response occurred to give small arteriole dilation and large arteriole constriction while venous diameters did not change. We conclude that: 1) changes in cardiac output during endotoxemia are not related to microvascular responses in skeletal muscle, and 2) the microvascular responses in skeletal muscle could be responsible for the decreased systemic vascular resistance during high cardiac output endotoxemia, but not for the elevated systemic vascular resistance during low cardiac output endotoxemia.

Serotonin-induced dilation of small arterioles is not mediated via endothelium-derived relaxing factor in skeletal muscle

Serotonin (5-HT) dilates precapillary arterioles in skeletal muscle. The purpose of this study was to determine if 5-HT releases endothelium-derived relaxing factor (EDRF) in this tissue. Diameters of third-order arterioles (A3) in the cremaster muscle of pentobarbital-anesthetized rats were measured via videomicroscopy. Concentration-response curves for acetylcholine, nitroprusside and 5-HT were obtained before and after the application of either hydroquinone (5 x 10(-4) M) or NG-nitro-L-arginine (10(-4) M). The involvement of prostaglandins was eliminated by ibuprofen (10(-4) M). In one group, 5-HT (20 micrograms/kg) and NG-nitro-L-arginine were given i.v. (30 mg/kg). The non-EDRF-dependent vasodilator papaverine (10(-5) M) was applied at the end of the protocol to determine the maximal resting diameter. When applied topically, both hydroquinone and NG-nitro-L-arginine significantly inhibited the dilation induced by acetylcholine, but neither agent affected the dilation to nitroprusside or 5-HT. NG-Nitro-L-arginine (i.v.) attenuated acetylcholine-induced dilation but not the dilation to intravenous 5-HT. These data suggest that 5-HT-induced dilation of small arterioles in skeletal muscle is EDRF-independent.

Effects of tissue acidosis on skeletal muscle microcirculatory responses to hemorrhagic shock in unanesthetized rats

Arteriolar dilatation, loss of venous tone, and uptake of shed blood characterize decompensated hemorrhagic shock. The loss of compensatory constrictor responses to hemorrhage mainly occurs in the skeletal muscle microcirculation. Tissue acidosis may be an important mediator of this phenomenon. Using a decerebrate in vivo rat cremaster muscle preparation, we observed the microcirculatory responses to hemorrhagic hypotension with cremaster bath conditions of pH 7.4 and pH 7.0. Our data indicate that tissue acidosis attenuates constrictor responses of larger arterioles (100-170 micron) and venules to hemorrhagic hypotension but has no effect on the dilator responses of small arterioles (10-30 micron). We conclude that tissue acidosis contributes significantly to loss of arteriolar resistance and to decreased venous return in the decompensatory phase of hemorrhagic shock.

Role of neutrophils on shock/resuscitation-mediated intestinal arteriolar derangements

Adequate resuscitation from hemorrhagic shock that preserves hemodynamics is associated with a generalized and progressive intestinal arteriolar vasoconstriction and hypoperfusion coupled with impairment of the endothelium-dependent dilation response. This study was performed to investigate the role of neutrophils on the postresuscitation intestinal arteriolar derangements. Experiments were performed in anesthetized rats 24 h after neutrophil depletion. Neutropenia was induced with antineutrophil serum by tail vein injection. Rats injected with rabbit serum lacking anti–rat neutrophil antibody served as controls. Hemorrhagic shock was 50% of mean arterial pressure for 60 min. Resuscitation was with the shed blood returned plus 2 volumes of saline. A nonhemorrhage group served as control. Intravital videomicroscopy of the terminal ileum was used to measure microvascular diameter and centerline red cell velocity. Endothelial function was assessed from the response to the endothelium-dependent dilator acetylcholine (10−9 to 10−4 M). Regardless of neutrophil count, hemorrhagic shock caused selective vasoconstriction of inflow A1 arterioles (−21.49 ± 0.67%) from baseline, which was not seen in the premucosal A3 vessels (pA3, dA3). At 2 h postresuscitation, there was a generalized vasoconstriction from baseline diameter in A1 (−21.26 ± 2.29%), pA3 (−22.66 ± 5.02%), and dA3 (−17.62 ± 4.84%). Neurophil depletion caused a significant reset of baseline A1 blood flow from 701 ± 90 nL/s to 978 ± 90 nL/s and attenuated the postresuscitation hypoperfusion. This occurred independently of the A1 diameter change. Hemorrhagic shock/resuscitation caused impairment of the endothelium-dependent dilation response irrespective of neutrophil count. This study demonstrates that neutrophils do not contribute to the hemorrhagic/resuscitation-mediated intestinal arteriolar derangements, but appear to possess a role in the intestinal arteriolar blood flow regulation under normal and low flow states possibly via a rheologic effect.

Progressive decrease in constrictor reactivity of the non-absorbing intestine during chronic sepsis.

Chronic sepsis leads to an impaired intestinal microcirculation, which might reflect altered microvascular control. We hypothesized that intestinal microvascular sensitivity to norepinephrine (NE) is decreased during chronic sepsis. Chronic sepsis was induced by a polymicrobial inoculation of implanted subcutaneous sponges in rats. Septic rats were studied either 24 or 72 h after a single inoculation (1-hit) of bacteria. Other rats received a second inoculation (2-hit) of bacteria 48 h later and were studied at 24 h after the second inoculation. NE (0.01-1.0 microM) responses in the non-absorbing terminal ileal arterioles (inflow A1, proximal-p and distal-d premucosal A3) were measured by video microscopy. NE threshold sensitivity (pD(T20) = -log of 20% response dose) was analyzed. pD(T20) was significantly decreased in A1, pA3, and dA3 of 1-hit 24-h septic rats (P < 0.05), and was further decreased in all vessels of 2-hit 72-h septic rats (P < 0.05). In contrast, the pDT(T20) of all three vessels significantly returned toward normal values after 72 h in rats that had only 1 bacteria inoculation. We conclude that an initial bacterial challenge decreases vasoconstrictor reactivity of the intestinal microcirculation and that subsequent repeated bacterial challenge exacerbates this defect in vasoconstrictor control in the non-absorbing intestine.

IMPAIRMENT OF ENDOTHELIUM-DEPENDENT DILATION RESPONSE AFTER RESUSCITATION FROM HEMORRHAGIC SHOCK INVOLVED POSTRECEPTOR MECHANISMS

Resuscitation from hemorrhagic shock is associated with impairment of the endothelium-dependent dilation response, whereas the dilation response induced by the endothelium-independent pathway, which is mediated by nitroprusside, a nitric oxide (NO) donor and a direct activator of guanylate cyclase, remains unaltered. Whether the impairment of the endothelium-dependent dilation response is caused by a specific receptor alteration or generally a defect in signal transduction pathway remains undetermined. Anesthetized rats were monitored for hemodynamics, and the terminal ileum was prepared for intravital videomicroscopy. Hemorrhage was 50% of mean arterial pressure for 60 min followed by resuscitation with the shed blood returned plus 2 volumes of normal saline. Intestinal microvascular reactivity to the endothelium-dependent receptor-dependent agonists acetylcholine or substance P (10−8 or 10−6 M), as well as the endothelium-dependent receptor-independent calcium ionophore, was determined at baseline and at 2 h postresuscitation from hemorrhagic shock. Measured vascular diameters for premucosal A3 arterioles (pA3 and dA3) were normalized and expressed as percentage of the maximal dilation capacity, as obtained from the response to the endothelium-independent NO donor sodium nitroprusside (10−4 M). At 2 h postresuscitation, there was a marked constriction of pA3 (−70.1 ± 20) and dA3 (−61.5 ± 11.6) from maximal dilation capacity. Baseline premucosal arteriolar response to substance P (10−8 M) was 30.68 ± 4.19% and 34.66 ± 5.82% for pA3 and dA3 arterioles, respectively. This was significantly reduced to 20.97 ± 2.41% and 17.94 ± 3.60% at 2 h postresuscitation. However, no significant difference between baseline and postresuscitation arteriolar responses was observed at the higher dose of substance P (10−6 M). Postresuscitation premucosal arteriolar response to the endothelium-dependent receptor-independent calcium ionophore (10−9 to 10−5 M) is characterized by a marked decrease in sensitivity and an enhanced threshold for calcium ionophore-mediated dilation. The logEC50 was −7.62 ± 0.39 and −7.75 ± 0.32 for the pA3 and dA3 at baseline, respectively. This was significantly (P < 0.01) reduced to −5.15 ± 0.14 and −4.39 ± 0.71 at 2 h postresuscitation. These data suggest that impairment of the endothelium-dependent dilation response after resuscitation from hemorrhagic shock is not mediated by specific receptor alteration. Cellular mechanisms that participate in or are part of oxygen free radical formation after resuscitation from hemorrhagic shock such as Ca2+ and leukocytes, appear to have a pivotal role in the mechanism of cellular dysfunction.