El Rasheid Zakaria

In vivo diffusion of immunoglobulin G in muscle: effects of binding, solute exclusion, and lymphatic removal

Previously, we demonstrated that immunoglobulin G (IgG), dissolved in an isotonic solution in the peritoneal cavity, transported rapidly into the abdominal wall when the intraperitoneal (ip) pressure was >2 cmH2O. We hypothesized that this was chiefly caused by convection and that diffusion of IgG was negligible. To investigate the role of diffusion, we dialyzed rats with no pressure gradient across the abdominal wall muscle for 2 or 6 h with an ip isotonic solution containing125I-labeled IgG. At the end of the experiment, the animal was euthanized and frozen and abdominal wall tissue was processed to produce cross-sectional autoradiograms. Quantitative densitometric analysis resulted in IgG concentration profiles with far lower magnitude than profiles from experiments in which convection dominated. In other in vivo experiments, we determined the lymph flow rate to be 0.8 × 10-4ml ⋅ min-1 ⋅ g-1and the fraction of extravascular tissue (θs) available to the IgG to be 0.041 ± 0.001. An in vitro binding assay was used to determine the time-dependent, nonsaturable binding constant: 0.0065 min-1 × duration of exposure. A non-steady-state diffusion model that included effects of θs, time-dependent binding, and lymph flow was fitted to the diffusion profile data, and the IgG diffusivity within the tissue void was estimated to be 2 × 10-7cm2/s, a value much higher than that published by other groups. We also demonstrate from our previous data that convection of IgG through tissue dominates over diffusion at ip pressures >2 cmH2O, but diffusion may not be negligible. Furthermore, nonsaturable binding must be accounted for in the interpretation of tissue protein concentration profiles.Previously, we demonstrated that immunoglobulin G (IgG), dissolved in an isotonic solution in the peritoneal cavity, transported rapidly into the abdominal wall when the intraperitoneal (ip) pressure was > 2 cmH2O. We hypothesized that this was chiefly caused by convection and that diffusion of IgG was negligible. To investigate the role of diffusion, we dialyzed rats with no pressure gradient across the abdominal wall muscle for 2 or 6 h with an ip isotonic solution containing 125I-labeled IgG. At the end of the experiment, the animal was euthanized and frozen and abdominal wall tissue was processed to produce cross-sectional autoradiograms. Quantitative densitometric analysis resulted in IgG concentration profiles with far lower magnitude than profiles from experiments in which convection dominated. In other in vivo experiments, we determined the lymph flow rate to be 0.8 x 10(-4) ml.min-1.g-1 and the fraction of extravascular tissue (theta s) available to the IgG to be 0.041 +/- 0.001. An in vitro binding assay was used to determine the time-dependent, nonsaturable binding constant: 0.0065 min-1 x duration of exposure. A non-steady-state diffusion model that included effects of theta s, time-dependent binding, and lymph flow was fitted to the diffusion profile data, and the IgG diffusivity within the tissue void was estimated to be 2 x 10(-7) cm2/s, a value much higher than that published by other groups. We also demonstrate from our previous data that convection of IgG through tissue dominates over diffusion at ip pressures > 2 cmH2O, but diffusion may not be negligible. Furthermore, nonsaturable binding must be accounted for in the interpretation of tissue protein concentration profiles.

In vivo effects of hydrostatic pressure on interstitium of abdominal wall muscle

Fluid loss from the peritoneal cavity to surrounding tissue varies directly with intraperitoneal hydrostatic pressure (Pip). According to Darcys law [Q = -KA(dPif/dx)], fluid flux (Q) across a cross-sectional area (A) of tissue will increase with an increase in either hydraulic conductivity (K) or the interstitial fluid hydrostatic pressure gradient (dPif/dx, where x is distance). Previously, we demonstrated that in the anterior abdominal muscle (AAM) of rats, dPif/dx increases by only 40%, whereas K rises fivefold between Pip of 1.5 and 8 mmHg. Because K is a function of interstitial volume (thetaif), we hypothesized that perturbations of Pip would change Pif and expand the interstitium, increasing thetaif. To test this hypothesis, we used dual-label quantitative autoradiography (QAR) to measure extracellular fluid volume (thetaec) and intravascular volume (thetaiv) in the AAM of rats within the Pip range from -2.8 to +8 mmHg. thetaif was obtained by subtraction (thetaec - thetaiv). dPif/dx was measured with a micropipette and a servo-null system. Local thetaiv did not vary with Pip and averaged 0.010 +/- 0.002 ml/g, and thetaif averaged 0. 19 +/- 0.01 ml/g at Pif </=1.2 mmHg. However, thetaif doubled between Pif of 1.2 and 4.2 mmHg (from 0.20 +/- 0.00 to 0.39 +/- 0.01 ml/g, respectively) but did not increase with further increases in Pif. This nonlinear pressure-volume relationship does not explain the fivefold increase in K with Pip. Because the interstitial matrix contributes to the interstitial resistance to fluid flow, and because hyaluronan (HA) is the only component of the matrix that is not anchored to the tissue, we hypothesized that the loss of interstitial HA was responsible for the continued decrease in interstitial resistance to fluid flow. We determined HA concentration in the rat AAM and adjacent subcutaneous tissue (SC) at Pip = 0 mmHg and after 2 h of dialysis at constant Pip = 6 mmHg. The HA content (normalized to dry weight) in the AAM was reduced from 487 +/- 16 to 360 +/- 27 micrograms/g dry tissue (n = 4, P < 0.05) and increased from 528 +/- 72 to 1,050 +/- 136 mg/g dry tissue (n = 4, P > 0.001) in the SC. We conclude that the mechanisms responsible for the increase in K with Pip include expansion of the interstitium, dilution of interstitial macromolecules, and washout from the AAM to SC of interstitial macromolecules responsible for resistance to fluid flow.

Intraperitoneal fluid volume changes during peritoneal dialysis in the rat: indicator dilution vs. volumetric measurements.

In order to validate the single injection RISA (125I human serum albumin) indicator diluation technique for assessing the alterations in intraperitoneal (i.p.) dialysate volume (IPV) which occur vs. time [V(t)] during peritoneal dialysis (PD), the RISA dilution technique was compared to V(t) determinations using a direct volume recovery method in Wistar rats. Sixteen milliliters of either 1.36 or 3.86% Dianeal or 0.9% NaCl were used as dialysis fluids in exchanges lasting between 1 and 360 min. Approximately 4% (4.41 +/- 0.59 (SE; n = 8) for 1.36% Dianeal and 4.07 +/- 0.72 (n = 4) for 3.86% Dianeal) of the RISA dose given intraperitoneally was lost from the dialysate during the first 1(-1.5) min after instillation, conceivably due to rapid tracer adsorption to peritoneal surfaces. Following the initial instant tracer loss and RISA dilution due to a residual volume (3.07 +/- 0.18 ml; n = 12), RISA disappeared at a fractional rate (FDR) of 2.10 +/- 0.14 x 10(-3) min-1 and 1.67 +/- 0.09 x 10(-3) min-1, during the first 30 min for 1.36 and 3.86% Dianeal, respectively. The overall FDR was 1.33 +/- 0.10 x 10(-3) and 0.707 +/- 0.082 x 10(-3) min-1 for 1.36% Dianeal (0-150 min) and 3.86% Dianeal (0-360 min), respectively, while the overall (0-150 min) FDR for the NaCl exchanges was 1.40 +/- 0.21 x 10(-3) min-1. These values correspond to RISA clearances out of the peritoneal cavity (KE) of 29.2 +/- 1.8, 22.1 +/- 1.6, and 25.7 +/- 2.4 microliter x min-1 for 1.36 and 3.86% Dianeal and 0.9% NaCl, respectively. The KE value for 3.86% Dianeal was significantly (p < 0.05) lower than for the two dialysates with lower osmolality. The slightly enhanced FDR of RISA during the first 30 min was partly due to the presence of nonprotein-bound free iodine in the RISA preparation used, and also to an enhanced disappearance of albumin during the first portion of the dwell. V(t) data from individual experiments using the RISA dilution technique (RISA curves) were analyzed by computer-aided nonlinear least-squares regression analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

In vivo hydraulic conductivity of muscle: effects of hydrostatic pressure

We and others have shown that the loss of fluid and macromolecules from the peritoneal cavity is directly dependent on intraperitoneal hydrostatic pressure (Pip). Measurements of the interstitial pressure gradient in the abdominal wall demonstrated minimal change when Pip was increased from 0 to 8 mmHg. Because flow through tissue is governed by both interstitial pressure gradient and hydraulic conductivity ( K), we hypothesized that K of these tissues varies with Pip. To test this hypothesis, we dialyzed rats with Krebs-Ringer solution at constant Pip of 0.7, 1.5, 2.2, 3, 4.4, 6, or 8 mmHg. Tracer amounts of125I-labeled immunoglobulin G were added to the dialysis fluid as a marker of fluid movement into the abdominal wall. Tracer deposition was corrected for adsorption to the tissue surface and for local loss into lymphatics. The hydrostatic pressure gradient in the wall was measured using a micropipette and a servo-null system. The technique requires immobilization of the tissue by a porous Plexiglas plate, and therefore a portion of the tissue is supported. In agreement with previous results, fluid flux into the unrestrained abdominal wall was directly related to the overall hydrostatic pressure difference across the abdominal wall (Pip = 0), but the interstitial pressure gradient near the peritoneum increased only ∼40% over the range of Pip = 1.5-8 mmHg (20-28 mmHg/cm). K of the abdominal wall varied from 0.90 ± 0.1 × 10-5cm2 ⋅ min-1 ⋅ mmHg-1at Pip = 1.5 mmHg to 4.7 ± 0.43 ×10-5cm2 ⋅ min-1 ⋅ mmHg-1on elevation of Pip to 8 mmHg. In contrast, for the same change in Pip, abdominal muscle supported on the skin side had a significantly lower range of fluid flux (0.89-1.7 × 10-4vs. 1.9-10.1 × 10-4ml ⋅ min-1 ⋅ cm-2in unsupported tissue). The differences between supported and unsupported tissues are likely explained in part by a reduced pressure gradient across the supported tissue. In conclusion, the in vivo hydraulic conductivity of the unsupported abdominal wall muscle in anesthetized rats varies with the superimposed hydrostatic pressure within the peritoneal cavity.

Mechanisms of Direct Peritoneal Resuscitation–Mediated Splanchnic Hyperperfusion Following Hemorrhagic Shock

Conventional resuscitation (CR) from hemorrhagic shock causes a persistent and progressive splanchnic vasoconstriction and hypoperfusion despite hemodynamic restoration with intravenous fluid therapy. Adjunctive direct peritoneal resuscitation (DPR) with a clinical peritoneal dialysis solution instilled into the peritoneal cavity has been shown to restore splanchnic tissue perfusion, down-regulate the gut-derived exaggerated systemic inflammatory response, promote early fluid mobilization, and improve overall outcome. This study was conducted to define the molecular mechanisms of DPR-induced gut hyperperfusion after hemorrhagic shock. Male rats were bled to 50% baseline mean arterial pressure and resuscitated with the shed blood plus two volumes of saline (CR). In vivo videomicroscopy and Doppler velocimetry were used to assess terminal ileal microvascular diameters and blood flow. Direct peritoneal resuscitation animals received CR and topical application of a clinical glucose-based peritoneal dialysis solution (Delflex). Inhibitors, glibenclamide (K+ATP channels), N-monomethyl-L-arginine (L-NMMA) (nitric oxide synthase), 8-cyclopentyl-1,3-diprophylxanthine (DPCPX) (A1 adenosine receptor), tetrabutylammonium (K+Ca2+ channels), and mefenamic acid (cyclooxygenase) were topically applied (individually or in combination) with DPR according to protocol; BQ-123 (endothelin A receptor antagonist) and BQ-788 (endothelin B receptor antagonist) were used topically with CR to define the mechanism of post-CR vasoconstriction and hypoperfusion. Conventional resuscitation caused a persistent progressive intestinal vasoconstriction and hypoperfusion that can be abolished with endothelin antagonists. In contrast, adjunctive DPR caused an instantaneous sustained vasodilation and hyperperfusion. Glibenclamide or L-NMMA partially attenuated DPR-induced vasodilation, whereas the addition of DPCPX to the two inhibitors eliminated the dilation. Cyclooxygenase and K+Ca2+channels were not active in DPR-mediated microvascular effects. In conclusion, DPR improves splanchnic tissue perfusion by endothelium-dependent mechanisms mediated by activations of glibenclamide-sensitive K+ channels (KATP), adenosine A1 receptor subtype activation, and nitric oxide release. Direct peritoneal resuscitation preserves endothelial dilatory functions, thereby overriding any endothelium-derived constrictor response triggered by hemorrhagic shock and CR.

Hemorrhagic shock and resuscitation-mediated tissue water distribution is normalized by adjunctive peritoneal resuscitation.

BACKGROUND Adjunctive direct peritoneal resuscitation (DPR) from hemorrhagic shock (HS) improves intestinal blood flow and abrogates postresuscitation edema. HS causes water shifts as a result of sodium redistribution and changes in transcapillary Starling forces. Conventional resuscitation (CR) with crystalloid aggravates water sequestration. We examined the compartment pattern of organ tissue water after HS and CR, and modulation of tissue edema by adjunctive DPR. STUDY DESIGN Rats were hemorrhaged (40% mean arterial pressure for 60 minutes) and assigned to four groups (n = 7): sham, no HS; HS no resuscitation; HS+CR (shed blood plus 2 volumes Ringers lactate); and HS+CR+DPR (20 mL clinical intraperitoneal (IP) dialysis fluid). Isotopic markers determined equilibrium distribution volumes [V(D)] in gut, liver, lung, and muscle by quantitative autoradiography (2-hour postresuscitation). Total tissue water (TTW) was determined by wet-dry weights. Extracellular water was measured from (14)C-mannitol V(D), and intravascular volume (IVV) from (131)I-labeled IgG V(D). Cellular and interstitial water volumes were calculated. RESULTS HS alone decreased IVV in all tissues and TTW in gut, lung, and muscle, but not liver, compared with shams. IVV remained decreased with all resuscitations despite restoration of central hemodynamics. CR caused interstitial edema in gut, liver, and muscle, and cellular edema in lung. DPR reduced (liver, muscle) or prevented (gut, lung) these volume shifts. CONCLUSIONS HS decreases IVV. HS-induced water shifts are organ-specific and prominent in gut, lung, and muscle. CR restores central hemodynamics, does not restore IVV, and alters organ-specific TTW distribution. Adjunctive DPR with IP dialysis fluid normalizes TTW and water compartment distribution and prevents edema. Combined effect of DPR and intravascular fluid replacement appears to prevent global tissue edema and improve outcomes from HS.

Preservation of hepatic blood flow by direct peritoneal resuscitation improves survival and prevents hepatic inflammation following hemorrhagic shock.

Conventional resuscitation (CR) from hemorrhagic shock (HS) results in gut and liver hypoperfusion, organ and cellular edema, and vital organ injury. Adjunct direct peritoneal resuscitation (DPR) with dialysate prevents gut vasoconstriction, hypoperfusion, and injury. We hypothesized that DPR might also improve hepatocellular edema, inflammation, and injury. Anesthetized male SD rats were assigned to groups (n = 8/group): 1) sham (no HS); 2) HS (40% MAP/60 min) + intravenous fluid conventional resuscitation [CR; shed blood + 2 vol saline (SAL)/30 min]; 3) HS+CR+DPR (30 ml ip 2.5% glucose dialysate); or 4) HS+CR+SAL (30 ml ip saline). Histopathology showed lung and liver injury in HS+CR and HS+CR+SAL up to 24-h postresuscitation (post-RES) that was not in shams and which was prevented by adjunct DPR. Wet-to-dry weight ratios in HS+CR revealed organ edema formation that was prevented by adjunct DPR. HS+CR and HS+CR+SAL had 34% mortality by 24-h post-RES, which was absent with DPR (0%). Liver IFN-γ and IL-6 levels were elevated in CR compared with DPR or shams. TNF-α mRNA was upregulated in CR/sham and DPR/sham. IL-17 was downregulated in DPR/sham. CXCL10 mRNA was upregulated in CR/sham but downregulated in DPR/sham. Despite restored central hemodynamic performance after CR of HS, liver blood flow was compromised up to 24 h post-RES, and the addition of DPR restores and maintains liver perfusion at 24-h post-RES. DPR prevented liver injury, histological damage, and edema formation compared with CR alone. DPR provided a mitigating anti-inflammatory dampening of the systemic inflammatory response. In all, these effects likely account for improved survivorship in the DPR-treated group.

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.