Pathophysiology of congenital diaphragmatic hernia. IV: Renal hyperplasia is associated with pulmonary hypoplasia.

The hypothesis of this article is that growth of the fetal lung is stimulated by a pulmonary growth factor (PGF) produced by the kidneys, which is modulated by a feedback signal from the lungs, a pulmonary-derived renotropin (PDR). In the fetus with pulmonary hypoplasia (PH), the lungs may maximally stimulate this feedback loop to release more PDR, resulting in continual stimulation of the kidneys and renal enlargement. If such a schema plays a role in the pathophysiology of PH, newborn infants with congenital diaphragmatic hernia (CDH) or chronic amniotic fluid leak (CAFL) should have enlarged kidneys. To investigate this hypothesis, we created models of CDH in fetal lambs and CAFL in fetal rabbits, and then analyzed lung (Lu) and kidney (K) growth. When compared to controls, newborn CDH lambs had significantly smaller lungs and larger kidneys. The lungs were hypoplastic as defined by either decreased lung weight/body weight (LuW/BW), lung DNA/body weight (Lu DNA/BW), or lung total protein/body weight (LuTP/BW) (P < .01). Renal hyperplasia was confirmed by KW/BW, K DNA/BW (P < .01), and KTP/BW (P < .05). An inverse relationship between lung size and kidney size could be described by the equation KW/BW = 1.04 - 0.12 LuW/BW (r = -.75). The CAFL model in newborn rabbits produced severe oligohydramnios when compared with controls (P < .01). This resulted in fetuses with smaller lungs and larger kidneys as compared with those of controls. The lungs were significantly smaller and more hypoplastic than controls when compared by LuW (P < .01), LuW/BW (P < .01), Lu DNA/BW (P < .05), and Lu TP/BW (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Combined preoperative and postoperative immunotherapy for murine C1300 neuroblastoma.

Preoperative treatment of murine C1300-neuroblastoma (C1300) with triple immunotherapy using low-dose cyclophosphamide (CY), retinyl palmitate (RP), and interleukin-2 (IL2), followed by tumor resection leads to significant initial tumor control and prolonged survival. However, because long-term tumor recurrence is 67%, the efficacy of continued postoperative immunotherapy is now evaluated. Thirty-two A/J mice with 1 cm subcutaneous C1300 tumors were treated for 13 days with CY-100 mg/kg, intraperitoneally (IP), on day 2 of treatment then 25 mg/kg on day 9, RP-2500 IU IP 2 x/week, and IL2 1.6 x 10(5) U IP BID on days 4 to 9 and 11 to 13. On day 14, mice were divided into five treatment groups: (1) OP (operated-tumor resection, n = 6); (2) OP+CY (resection and postoperative CY, n = 7); (3) OP+CY+RP (resection and postoperative CY+RP, n = 7); (4) OP+CY+RP+IL2 (resection and postoperative CY+RP+IL2, n = 7); and (5) CY+RP+IL2 (continued CY+RP+IL2 with no resection, n = 5). Survival and postoperative tumor recurrence were followed for 60 days. The cure rates were group 1 33% (2/6), group 2 43% (3/7), group 3 29% (2/7), group 4 71% (5/7), and group 5 20% (1/5). After surgery, tumors that recurred did so in 8 to 22 days, with no statistical difference noted between groups. MHC class I antigenic expression of tumors resected on day 14 and recurrent tumors was determined with monoclonal antibodies and flow cytometry. In tumors resected on day 14, class I expression measured by mean fluorescence, was 374.8 +/- 27.40.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathophysiology of congenital diaphragmatic hernia. X: Localization of nitric oxide synthase in the intima of pulmonary artery trunks of lambs with surgically created congenital diaphragmatic hernia.

The pathophysiology of congenital diaphragmatic hernia (CDH) results from a combination of pulmonary hypoplasia, pulmonary hypertension, and surfactant deficiency. Previously we demonstrated that inhaled nitric oxide (NO), a known vasodilator, only improves oxygenation and decreases pulmonary artery pressures when the lamb model of CDH is pretreated with exogenous surfactant. Nitric oxide synthase (NOS) in endothelial cells is responsible for the production of NO, a mediator of smooth muscle cell relaxation. Pulmonary hypertension in CDH may result from a defect in the endogenous production of NO. Our aim was to determine whether the main pulmonary artery trunks in CDH lambs have NOS immunoreactivity. Cryostat sections of paraformaldehyde-fixed specimens of pulmonary artery and aortic rings from 10 CDH lambs and five control lambs were processed for NADPH-diaphorase activity. Immunolocalization of NOS was studied in paraformaldehyde-fixed sections and compared with serially cut specimens from identical rings that were tested for NADPH-diaphorase activity. Intense NADPH-diaphorase staining was present in the intimal layer (endothelial lining) of the pulmonary artery and aortic rings of both the CDH and control lambs. This activity colocalized with NOS immunoreactivity in all specimens. Both NOS immunoreactivity and NADPH-diaphorase staining were lacking in cartilage, which were used as negative controls. NOS is present in the main pulmonary artery trunks of CDH lambs. To our knowledge, this is the first report of NOS immunoreactivity in CDH. We can only speculate whether this activity is preserved in other areas of the vascular tree in CDH, ie, pulmonary capillaries and veins. Perhaps the pulmonary hypertension in CDH is not caused by an NOS deficiency.

Oxygenated intraluminal perfluorocarbon protects intestinal muscosa from ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury to the intestinal mucosa occurs in several commonly encountered clinical situations, such as necrotizing enterocolitis and nonocclusive mesenteric insufficiency. No clinically feasible technique is available for mucosal preservation during ischemia. The goal of this work was to determine whether the continuous intraluminal flow of oxygenated perfluorocarbon (PFC) could protect mucosal integrity and function in a rat model of intestinal I/R injury. Rats were subjected to ischemia by clipping the superior mesenteric artery (SMA) for 60 minutes. Reperfusion was achieved by release of the clip for 120 minutes. Animals were divided into 4 groups: Sham (laparotomy alone), I/R (I/R alone), I + PFC/R (PFC was administered during the ischemic interval only), I/R + PFC (PFC was delivered only during reperfusion). Tissue sections were examined blindly to assess mucosal integrity, and mucosal dissacharidase activities were measured to assess function. Oxygenated PFC, when administered during ischemia alone, ameliorated I/R-induced mucosal injury; however, when it was delivered during reperfusion alone, the mucosal injury worsened. When oxygenated PFC was administered throughout I/R, the degree of mucosal injury was similar to the I + PFC/R group, and dissacharidase activities were preserved when compared with the I/R group. Intraluminal perfusion of oxygenated PFC during ischemia preserves mucosal function and integrity, and may offer a new treatment modality for a variety of mesenteric ischemic disorders.

Neutrophil activation and chemotaxis after in vitro treatment with perfluorocarbon.

The in vitro effects of perfluorocarbon (PFC) on human neutrophil activation and chemotaxis were examined. Neutrophils were incubated, with and without PFC, and were analyzed for chemotaxis through 5-microns and 8-microns pore filters. Neutrophil activation was quantitated by flow cytometry. Activation studies showed that PFC-treated neutrophil samples (n = 6) produced only 39.83% +/- 25.9% (mean +/- SD) of matched control (n = 6) fluorescence. Chemotaxis studies showed that PFC-treated neutrophil samples (n = 8) had migration of only 18.63% +/- 6.5% (of control values) of neutrophils to the outer boundary of the 5-microns filters (n = 8 controls). The 8-microns pore filter migration (n = 8) was similarly low, with a mean outer boundary migration count of only 26% +/- 19.8% of the control (n = 8) value. Thus, neutrophils exposed to perfluorocarbon produce significantly less detectable H2O2 (P < .001) and have a significantly lower chemotactic response (P < .001).

Postoperative immunotherapy of murine C1300-neuroblastoma.

Low-dose cyclophosphamide (CY) is an immunomodulating agent that down-regulates T suppressor cell function. This study investigates postoperative immunotherapy with CY as an alternate treatment for advanced immunogenic tumors such as neuroblastoma that typically respond poorly to traditional high-dose chemotherapy. A/J mice with 1.5-cm subcutaneous C1300-neuroblastoma (C1300-NB) tumors were divided into the following treatment groups: I, untreated (n = 14); II, 85% tumor resection (n = 18); III, sham-operated (n = 18); IV, multiple-dose CY (n = 6); V, 85% resection and single-dose CY (n = 14); VI, 85% resection and multiple-dose CY (n = 14). CY (100 mg/kg, intraperitoneally) was given initially 24 hours post-operatively to groups IV, V, and VI. Groups IV and VI also received weekly maintenance doses of 25 mg/kg CY. Results showed significantly increased survival (log-rank test) in CY-treated groups (IV, V, VI) compared with control groups (I,II,III). Cures were observed only in groups receiving partial resection plus CY (V, 7%; VI, 29%). Although surgical debulking of tumor alone (II) did not enhance survival, the procedure normalized depressed total lymphocyte counts and the subpopulation of Lyt 2,3+ (T suppressor/cytolytic cells) in the immediate postoperative period during which immunotherapy with CY was instigated. This may have contributed to the success of CY immunotherapy. To characterize the tumor-host immune interaction, additional studies were performed. Results showed the following. (1) Mice cured by debulking plus CY (from groups V and VI) could not be successfully reimplanted with C1300-NB, demonstrating immunologic mediation by CY.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathophysiology of congenital diaphragmatic hernia. VIII: Inhaled nitric oxide requires exogenous surfactant therapy in the lamb model of congenital diaphragmatic hernia.

The pathophysiology of the lamb model of congenital diaphragmatic hernia (CDH) involves pulmonary hypoplasia, pulmonary hypertension, and surfactant deficiency. Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. The aim of this study was to determine the effects of inhaled NO on pulmonary gas exchange, acid-base balance, and pulmonary pressures in a lamb model of CDH with or without exogenous surfactant therapy. At the gestational age of 78 days (full term, 145 days) 11 lamb fetuses had a diaphragmatic hernia created via a left thoracotomy and then were allowed to continue development in utero. After cesarean section, performed at term, six lambs received exogenous surfactant therapy (50 mg/kg, Infasurf) and five served as controls. All animals were pressure-ventilated for 30 minutes and then received 80 ppm of inhaled NO at an F1O2 of .9 for a 10-minute interval. Compared with the control lambs, the lambs with exogenous surfactant therapy had higher pH (7.17 +/- .06 v 6.96 +/- .07; P < .05), lower PCO2 (73 +/- 8 v 122 +/- 20, p < .05), and higher PO2 (153 +/- 38 v 50 +/- 23; P < .05). In control CDH lambs (without surfactant), inhaled NO did not improve pH, PCO2, or PO2, or decrease pulmonary artery pressure. In CDH lambs given exogenous surfactant, NO decreased pulmonary artery pressures (42 +/- 4 v 53 +/- 5; P < .005) and further improved PCO2 and PO2. NO also made the difference between pulmonary and systemic artery pressures more negative in the surfactant-treated lambs (-15 +/- 4 v -2.3 +/- 2.4; P < .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced resection and improved survival in murine neuroblastoma (C1300-NB) after preoperative immunotherapy.

Advanced neuroblastoma treated with standard chemotherapy has a poor prognosis. Combination immunotherapy for murine neuroblastoma with retinyl palmitate, low-dose cyclophosphamide, and interleukin-2 resulted in increased survival, impaired tumor growth, easier surgical resection, and increased class I expression or tumor cells. Preoperative immunotherapy may be useful in treatment of advanced human neuroblastoma.

Pathophysiology of congenital diaphragmatic hernia. XI: Anatomic and biochemical characterization of the heart in the fetal lamb CDH model.

AIM: The purpose of this study was to determine whether the presence of bowel in the chest during development in the fetal lamb model of congenital diaphragmatic hernia (CDH) results in structural and/or biochemical hypoplasia of the left venticle. METHODS: The model was created at 80 days' gestation and delivered at term. The hearts were fixed in 4% formaldehyde solution, components weighed, and right ventricular (RV) and left ventricular (LV) wall thicknesses and both aortic (Ao) and pulmonary artery (PA) root diameters were measured. Fresh specimens were analyzed for protein, DNA, hydroxyproline, and elastin content. All CDH measurements are compared with littermate control tissues. RESULTS: There were no differences in body weight (kg) between CDH and control littermates (4.25 +/- 0.26 versus 3.71 +/- 0.24, P = NS). CDH lambs have significantly decreased total heart (4.88 +/- .25 versus 6.75 +/- .49, P < .05), left ventricular (1.65 +/- .11 versus 2.15 +/- .19, P < .05), septal (1.29 +/- .11 versus 1.99 +/- .21, P < .05), and combined atrial (0.68 +/- .06 versus 1.14 +/- .15, P < .05) weights (g/kg lamb) without differences in RV weights (1.26 +/- .07 versus 1.57 +/- .17, P = NS). LV and RV wall thickness, and Ao root diameters (cm) were found to be identical in both CDH and control lambs. However, PA root diameters (0.47 +/- .01 versus 0.38 +/- .01, P < .005) and ductus arteriosus diameters were increased in CDH (0.35 +/- .01 versus 0.22 +/- .02, P < .005). Total protein, DNA collagen, and elastin content and DNA/total protein ratios were identical in RV and LV in both CDH and control lambs. CONCLUSION: Newborn lambs with left-sided CDH have a significantly lower total heart, LV, septal, and atrial weights without differences of RV weight or ventricular wall thicknesses. Given these findings, the unchanged DNA/protein ratio implies that the left ventricle is hypoplastic in CDH. Ao/PA root ratios suggest that LV hypoplasia in utero may result in increased left atrial pressures, decreased right-to-left shunting through the foramen ovale, and increased PA pressures and flow, resulting in increased PA root and ductus arteriosus diameters. This model simulates the clinical data from human fetuses/neonates with CDH. Further investigations are necessary to determine the functional significance of these findings.

Pathophysiology of congenital diaphragmatic hernia. XVI: Elevated pulmonary collagen in the lamb model of congenital diaphragmatic hernia.

The pathophysiologic features of congenital diaphragmatic hernia (CDH) include pulmonary hypoplasia, pulmonary hypertension, surfactant deficiency, and decreased pulmonary compliance. When the surfactant deficiency is corrected using exogenous surfactant therapy, the pulmonary compliance improves, but does not reach normal values. Quasistatic saline pressure-volume measurements, which eliminate the air-liquid interface, confirm that CDH lungs are intrinsically less compliant than control lungs. The authors hypothesized that this abnormal lung compliance results from elevated concentrations of collagen and/or elastin in the lung. Therefore, they measured the collagen and elastin concentrations in CDH and control lung tissue. Also measured was the collagen concentration in the kidney, intestine, and dissected third-generation arterioles, venules, and bronchioles, to characterize further the pathology of CDH. The CDH model was created on the left side of fetuses in pregnant ewes at 80 days' gestation. The fetuses were delivered and killed at 140 days (full term, 145). The concentrations of collagen (as hydroxyproline), elastin, DNA, and total protein were measured using standard techniques. Although there was significantly more collagen per gram of lung tissue in the CDH lungs (1.334 mg/g v 0.885 mg/g in the controls) the elastin concentrations were not different. The elevated collagen concentration was not associated specifically with the conducting airways or vasculature. The collagen concentrations in CDH kidneys and intestines were the same as those of controls. The DNA/total protein ratios in the CDH and control lungs were identical. The results suggest that the elevated collagen concentration was present only in the lungs of CDH lambs, and that it was not attributable to atrophy or hypertrophy of the lungs. Thus, increased collagen in the lung parenchyma may be responsible for the intrinsic stiffness and decreased compliance of the CDH lungs.

Attenuated nitric oxide synthase activity and protein expression accompany intestinal ischemia/reperfusion injury in rats.

Intestinal ischemia/reperfusion (I/R) leads to bowel impairment via the release of reactive oxygen species (ROS) and neutrophil infiltration. In addition to modulating intestinal integrity, nitric oxide (NO(*)) inhibits neutrophil activation and scavenges ROS. Attenuated endogenous NO(*) formation may result in the accrual of these deleterious stimuli. Therefore, we determined nitric oxide synthase (NOS) activity in anesthetized rats subjected to 1 h of superior mesenteric ischemia or ischemia followed by reflow. NOS activity was measured in intestinal tissue homogenates as the conversion rate of (3)H-L-arginine to (3)H-L-citrulline. Our results demonstrate that intestinal ischemia leads to a decrease in NOS activity indicating lower NO(*) formation in the animal model. The attenuation in NOS activity was not reversed following 4 h of reperfusion. Western blot analysis revealed that the decline in enzyme activity was accompanied by reduced intestinal NOS III (endothelial constitutive NOS) expression. These findings provide biochemical evidence for impaired NO(*) formation machinery in intestinal I/R injury.

Intestinal and hemodynamic impairment following mesenteric ischemia/reperfusion.

BACKGROUND: Clinical intestinal ischemia/reperfusion (I/R) injury results in local and systemic dysfunction. A rat model of transient mesenteric occlusion has been used to study this phenomenon. However, a systematic analysis of the rat model with respect to intestinal permeability and hemodynamics has not been carried out. MATERIALS AND METHODS: In anesthetized rats, the superior mesenteric artery was occluded for 60 min, followed by reperfusion for 4 h. Intestinal impairment was evaluated via histological examination and by measuring ex vivo apparent permeability coefficients (Papp) of mannitol (0.18 kDa), inulin (5 kDa), and dextran (70 kDa). Hemodynamic effects of intestinal I/R were determined by monitoring mean arterial pressure (MAP) and heart rate (HR) via a catheter placed in the femoral artery. RESULTS: The animal model was associated with increased ex vivo Papp for mannitol and inulin. Although I/R injury was accompanied by significant histological disruption, there was no observable alteration in dextran permeability, suggesting that the loss in normal barrier function was limited to low-molecular-weight compounds. Hemodynamic measurements indicated that reperfusion induced a precipitous and sustained fall in MAP. HR values fell sharply following reperfusion but gradually increased and eventually "overshot" to values greater than baseline. CONCLUSIONS: Our findings demonstrate the selective loss of barrier function of the small bowel following intestinal I/R. Furthermore, these results also illustrate the importance of selecting appropriate permeability markers for the evaluation of intestinal damage. In light of the significant hemodynamic disruption accompanying the animal model, our investigation also points toward the need for developing therapeutic strategies that mitigate the local and systemic effects of intestinal I/R injury.

Mucosal protection from intestinal ischemia-reperfusion reduces oxidant injury to the lung.

The authors investigated whether amelioration of intestinal mucosal injury, due to ischemia-reperfusion (I/R), with oxygenated perfluorocarbon (PFC) would reduce an oxidant-generated lung injury. The small intestine is increasingly recognized as a primary effector of distant organ injury. Clinical and experimental studies suggest oxidant species and activated neutrophils as the agents responsible for lung injury after intestinal I/R. The role of intestinal mucosal injury has not been defined. Oxygenated PFC was perfused through the lumen of the intestine during periods of I/R. Portal venous effluent was examined for reactive oxygen species and lung tissue was examined for lipid peroxidation. Luminal perfusion of oxygenated PFC during intestinal I/R reduced oxidant species in the portal blood. This correlated with a reduction in lung lipid peroxidation. Oxygenated PFC prevented intestinal mucosal injury resulting from induced I/R. Amelioration of mucosal injury reduced oxidant generation in the portal venous circulation that was proportional to the reduction in measured lung injury. Protection of the mucosa with intraluminal oxygen may prevent I/R-associated lung injury.