Calculating postnatal growth velocity in very low birth weight (VLBW) premature infants.

OBJECTIVE: Currently, there is no standardized approach to the calculation of growth velocity (GV; g kg (-1) day(-1)) in hospitalized very low birth weight (VLBW) infants. Thus, differing methods are used to estimate GV, resulting in different medical centers and studies reporting growth results that are difficult to compare. The objective of this study was to compare actual GV calculated from infant daily weights during hospitalization in a Neonatal Intensive Care Unit (NICU) with estimated GV using two mathematical models that have been shown earlier to provide good estimated GVs in extremely low birth weight (ELBW) infants: an exponential model (EM) and a 2-Point model (2-PM). STUDY DESIGN: Daily weights from 81 infants with birth weights (BWs) of 1000 to 1499 g were used to calculate actual GV in daily increments from two starting points: (1) birth and (2) day of life (DOL) of regaining BW. These daily GV values were then averaged over the NICU stay to yield overall NICU GV from the two starting points. We compared these actual GV with estimated GV calculated using the EM and 2-PM methods. RESULTS: The mean absolute difference between actual and EM estimates of GV showed <1% error for 100% of infants from both starting points. The mean absolute difference between actual and 2-PM estimates showed <1% error for only 38 and 44% of infants from birth and regaining BW, respectively. The EM was unaffected by decreasing BW and increasing length of NICU stay, whereas the accuracy of the 2-PM was diminished significantly (P<0.001) by both factors. CONCLUSION: In contrast to the 2-PM, the EM provides an extremely accurate estimate of GV in larger VLBW infants, and its accuracy is unaffected by common infant factors. The EM has now been validated for use in all VLBW infants to assess growth and provides a simple-to-use and consistent approach.

Ursodeoxycholic acid ameliorates ibuprofen-induced enteropathy in the rat.

BACKGROUND: The objective of the study was to determine whether ursodeoxycholic acid (Ursodiol) is protective against ibuprofen (IBU)-induced enteropathy. METHODS: Using the chronically catheterized rat model, IBU (60 mg/kg body weight per day) was infused via the gastric catheter twice daily. Pancreatic enzyme (PE; 10,000 U lipase/kg body weight per day) and Ursodiol (10 mg/kg body weight per day) in two doses were infused via the duodenal catheter. Rats were assigned to one of six treatment groups and were administered treatment for 20 days: control, IBU, PE, IBU + PE, IBU + Ursodiol, and IBU + PE + Ursodiol. The entire jejunum, ileum, cecum, and colon were available for histologic analysis using previously described techniques. RESULTS: Addition of Ursodiol to high-dose IBU and normal doses of PE showed a significant reduction in the percentage of rats with ulcers (P < 0.05), total number of serositis events (P < 0.01), total number of severe ulcers (P < 0.001), and an absence of ulcers in the large intestine. CONCLUSIONS: Ursodiol, the drug of choice for the treatment of cystic fibrosis liver disease, may offer a safe method of using high-dose IBU in these patients by ameliorating the enteropathy.

TNF-alpha increases sensitivity to LPS in chronically catheterized rats.

Patients with severe trauma injury are transiently exposed to increased serum concentrations of tumor necrosis factor-alpha (TNF-alpha). These patients are susceptible to the development of multisystem organ failure (MSOF) triggered by subsequent exposure to bacterial toxins either via infection or increased intestinal permeability. We simulated the cytokine response of trauma by infusing 0.8 or 8.0 microg/kg of TNF-alpha (priming dose) into chronically catheterized rats. After 48 h, rats were challenged with endotoxin [lipopolysaccharide (LPS); 10 or 1,000 microg/kg]. Animals primed with either dose of TNF-alpha and then challenged with 1,000 microg/kg of LPS demonstrated significantly increased mortality, mean peak serum concentrations of interferon-gamma (IFN-gamma), and blood lactate concentrations (P < 0.05) compared with nonprimed animals. Mean peak serum concentrations of IFN-gamma and blood lactate concentrations were increased after challenge with 10 microg/kg of LPS only in animals primed with 8.0 microg/kg of TNF-alpha. Priming with TNF-alpha did not increase mortality after challenge with 10 microg/kg of LPS. These data suggest that both TNF-alpha release and the subsequent exposure to bacterial toxins mediate the pathophysiological progression from trauma to subsequent MSOF.

Endotoxin-induced reduction in biliary indocyanine green excretion rate in a chronically catheterized rat model.

Using a nonstressed chronically catheterized rat model in which the common bile duct was cannulated, we studied endotoxin-induced alterations in hepatic function by measuring changes in the maximal steady-state biliary excretion rate of the anionic dye indocyanine green (ICG). Biliary excretion of ICG was calculated from direct measurements of biliary ICG concentrations and the bile flow rate during a continuous vascular infusion of ICG. Despite significant elevations in mean peak serum tumor necrosis factor-alpha (TNF-alpha) concentrations (90.9 +/- 16.2 ng/ml), there was no effect on mean rates of bile flow or biliary ICG clearance after administration of 100 microg/kg endotoxin at 6 or 24 h. Significant differences from mean baseline rates of bile flow and biliary ICG excretion did occur after administration of 1,000 microg/kg endotoxin (mean peak TNF-alpha 129.6 +/- 24.4 ng/ml). Furthermore, when rats were treated with up to 16 microg/kg of recombinant TNF-alpha, there was no change in mean rates of bile flow or ICG biliary clearance compared with baseline values. These data suggest that the complex regulation of biliary excretion is not mediated solely by TNF-alpha.

Staphylococcal enterotoxin B potentiates LPS-induced hepatic dysfunction in chronically catheterized rats.

Most models of liver dysfunction in sepsis use endotoxin (lipopolysaccharide; LPS) to induce a pathophysiological response. In our study published in this issue (Beno DWA, Uhing MR, Goto M, Chen Y, Jiyamapa-Serna VA, and Kimura RE. Am J Physiol Gastrointest Liver Physiol 280: G858-G865, 2001), the adverse effect of LPS on hepatic function in vivo was only significant at relatively high LPS doses despite high tumor necrosis factor-alpha concentrations. However, many patients with sepsis are exposed to multiple bacterial toxins that may augment the immune response, resulting in increased hepatic dysfunction. We have developed a model of polymicrobial sepsis by parentally administering a combination of staphylococcal enterotoxin B (SEB) and LPS. Using this model, we demonstrate that SEB (50 microg/kg) potentiates the effect of LPS-induced hepatic dysfunction as measured by decreased rates of biliary indocyanine green clearance and bile flow. These increases were most pronounced with doses of 10 and 100 microg/kg LPS, doses that by themselves do not induce hepatic dysfunction. This may explain the seemingly increased incidence and severity of liver dysfunction in sepsis, and it suggests that the exclusive use of LPS for replicating septic shock may not be relevant for studies of hepatic dysfunction.

Neonatal subglottic stenosis--incidence and trends.

OBJECTIVE/HYPOTHESIS: Neonatal subglottic stenosis is a known entity arising from endotracheal tube intubation. In the 1970s and 1980s, estimates of the incidence of subglottic stenosis were in the range of 0.9% to 8.3% of intubated neonates. Because of improved techniques of handling neonates who require ventilatory support, we thought the actual incidence of neonatal subglottic stenosis in the late 1990s was much lower. STUDY DESIGN: We retrospectively reviewed all neonatal intensive-care unit (NICU) admissions from 1997 at our institution, which serves as a level 3 NICU. We also performed a MEDLINE search of the reported incidence of neonatal subglottic stenosis between 1960 and 1999. METHODS: Analysis was performed to identify all children who developed subglottic stenosis at our institution. Data were also collected and analyzed with regard to average gestational age, average birth weight, average duration of intubation, and the number of children requiring tracheostomy. The reports identified in the literature were reviewed as to the incidence of subglottic stenosis. RESULTS: A total of 544 neonates were admitted to the unit. Of these, 281 were intubated for an average of 11 days. No patients developed subglottic stenosis. Three patients required tracheostomies for other reasons. All studies published after 1983 reported an incidence of neonatal subglottic stenosis as less than 4.0%, and all studies published after 1990 reported an incidence of neonatal subglottic stenosis as less than 0.63%. CONCLUSIONS: Although our report applies to only a single institution in a single year, after reviewing the literature we think a downward trend exists in the incidence of neonatal subglottic stenosis in the late 1990s. The current incidence of neonatal subglottic stenosis is likely between 0.0% and 2.0%.

The effect of surgical stress on the endotoxin-induced interferon-gamma response.

Most animal studies of cytokine release during sepsis or endotoxemia have used models in which studies are performed during or immediately after surgical stress. In a previous study, we showed that surgical stress as measured by elevated endogenous corticosterone concentrations attenuated the endotoxin-induced tumor necrosis factor alpha (TNFalpha) response. To determine whether surgical stress attenuates the endotoxin-induced interferon-gamma (IFN-gamma) response, chronically catheterized male Sprague-Dawley rats were treated with endotoxin 10 microg/kg immediately after surgery for catheter placement (surgical stress group, SS group) or at least 4 days postoperative (nonstressed group, NS group). We found that peak endotoxin-induced IFN-gamma responses were similar in the SS and NS groups (2094 +/- 315 pg/mL vs. 1863 +/- 307 pg/mL). Baseline corticosterone concentrations were significantly elevated in the SS group compared to the NS group (273.8 +/- 15.2 ng/mL vs. 30.0 +/- 8.5 ng/mL, P < 0.001). Peak TNFalpha concentrations were significantly reduced in the SS group compared to the NS group (5.2 +/- 1.9 ng/mL vs. 69.9 +/- 10.3 ng/mL, P = 0.0002). While peak serum TNFalpha concentrations were inversely related to baseline corticosterone concentrations, there was no correlation between peak IFN-gamma concentrations and baseline corticosterone concentrations or between TNFalpha and IFN-gamma concentrations. We conclude that surgical stress associated with elevated concentrations of endogenous corticosterone does not attenuate the endotoxin-induced IFN-gamma response despite an attenuation of the endotoxin-induced TNFalpha response. Because the effect of stress on different cytokines is varied, studies of sepsis and endotoxemia must account for the effects of experimentally-induced stress on cytokine responses.

The effects of high-dose ibuprofen and pancreatic enzymes on the intestine of the rat.

BACKGROUND: High-dose ibuprofen therapy limits the progression of lung disease in patients with cystic fibrosis. However, ibuprofen increases intestinal permeability, which potentiates intestinal damage caused by high-dose pancreatic enzyme treatment, as was shown in a previous study by this group. In the present study, the combined effects of ibuprofen and pancreatic enzyme treatment on the intestine and liver were examined. METHODS: Using a chronically catheterized rat model, high-dose ibuprofen (60 mg/kg x day in two doses), with or without pancreatic enzyme treatment was infused into gastric and duodenal catheters, respectively, for 20 days. Six groups were studied: control group; ibuprofen treatment alone; pancreatic enzyme treatment alone (two groups: normal dose, 10,000 U lipase/kg x day and high dose, 40,000 U lipase/kg x day); and ibuprofen combined with pancreatic enzyme (two groups: ibuprofen with high-dose pancreatic enzyme and ibuprofen and low-dose pancreatic enzyme). After treatment, rats were autopsied, and complete histologic analyses of the entire intestine and liver were performed. RESULTS: Ibuprofen caused mild ulceration of the small intestine in 50% of rats. Pancreatic enzyme treatment alone did not induce ulceration of the intestine. The combination of pancreatic enzyme and ibuprofen treatment increased the severity of the ulcers in the small intestine but not the number of ulcers or the percentage of rats affected. Ibuprofen treatment alone did not cause ulcers in the large intestine, but with the addition of pancreatic enzymes, ulceration and fibrosis were present. CONCLUSIONS: Ibuprofen at doses used to limit progression of cystic fibrosis lung disease caused enteropathy in 50% of rats. There was synergism between ibuprofen and pancreatic enzyme treatment in the production of severe ulcers. Ulcers in the cecum and colon were increased with combined ibuprofen and pancreatic enzyme treatment compared with incidence in control animals.

Nonstressed rat model of acute endotoxemia that unmasks the endotoxin-induced TNF-alpha response.

Previous investigators have demonstrated that the tumor necrosis factor-alpha (TNF-alpha) response to endotoxin is inhibited by exogenous corticosterone or catecholamines both in vitro and in vivo, whereas others have reported that surgical and nonsurgical stress increase the endogenous concentrations of these stress-induced hormones. We hypothesized that elevated endogenous stress hormones resultant from experimental protocols attenuated the endotoxin-induced TNF-alpha response. We used a chronically catheterized rat model to demonstrate that the endotoxin-induced TNF-alpha response is 10- to 50-fold greater in nonstressed (NS) rats compared with either surgical-stressed (SS, laparotomy) or nonsurgical-stressed (NSS, tail vein injection) models. Compared with the NS group, the SS and NSS groups demonstrated significantly lower mean peak TNF-alpha responses at 2 mg/kg and 6 micrograms/kg endotoxin [NS 111.8 +/- 6.5 ng/ml and 64.3 +/- 5.9 ng/ml, respectively, vs. SS 3.9 +/- 1.1 ng/ml (P < 0.01) and 1.3 +/- 0.5 ng/ml (P < 0.01) or NSS 5.2 +/- 3.2 ng/ml (P < 0.01) at 6 micrograms/kg]. Similarly, baseline concentrations of corticosterone and catecholamines were significantly lower in the NSS group [84.5 +/- 16.5 ng/ml and 199.8 +/- 26.2 pg/ml, respectively, vs. SS group 257. 2 +/- 35.7 ng/ml (P < 0.01) and 467.5 +/- 52.2 pg/ml (P < 0.01) or NS group 168.6 +/- 14.4 ng/ml (P < 0.01) and 1,109.9 +/- 140.7 pg/ml (P < 0.01)]. These findings suggest that the surgical and nonsurgical stress inherent in experimental protocols increases baseline stress hormones, masking the endotoxin-induced TNF-alpha response. Subsequent studies of endotoxic shock should control for the effects of protocol-induced stress and should measure and report baseline concentrations of corticosterone and catecholamines.

Indomethacin and pancreatic enzymes synergistically damage intestine of rats.

The use of high-dose pancreatic enzymes by patients with cystic fibrosis was associated with the development of fibrosing colonopathy. Preliminary studies indicated that the infusion of high-dose pancreatic enzymes alone did not cause intestinal damage. We hypothesized that cystic fibrosis patients that developed fibrosing colonopathy had increased intestinal permeability. Our goal was to develop a rat model for pancreatic enzyme-induced fibrosing colonopathy by increasing intestinal permeability with the use of indomethacin. Pancreatic enzymes, 150,000 units/kg/day, and indomethacin, 3 mg/kg/day, alone and in combination were administered via duodenal catheter to rats for 10 days. Indomethacin and pancreatic enzymes caused intestinal damage, resulting in significant increases in the total number of ulcers (P < 0.007), the number of severe ulcers (P < 0.003), and ulcers in the cecum and colon (P < 0.0007). We conclude that the combination of indomethacin and pancreatic enzymes acts synergistically to cause damage to the intestine.

The effect of intestinal permeability on pancreatic enzyme-induced enteropathy in the rat.

BACKGROUND: Fibrosing colonopathy in cystic fibrosis occurs in children 2 to 7 years old and is associated with excess doses of high and regular strength lipase pancreatic enzymes, given for more than 6 months. A rat model was developed to study the effects of excessive doses of pancreatic enzymes equivalent to those producing fibrosing colonopathy in cystic fibrosis patients. METHODS: Five groups of animals were studied after administration of different combinations of pancreatic enzymes, oleic acid, and reserpine. RESULTS: Pancreatic enzymes alone produced minimal damage to the intestine and none to the liver. Excessive doses of pancreatic enzymes in combination with agents that increased intestinal permeability (oleic acid, reserpine) were associated with intestinal eosinophilia and necrosis of the jejunoileal muscle layer and inflammatory nodules in the liver, which increased with duration of insult. CONCLUSIONS: Increased intestinal permeability potentiates damage to the intestine caused by excessive pancreatic enzyme dosage. It is a characteristic of cystic fibrosis that may increase vulnerability to fibrosing colonopathy.

Neonatal intestinal metabolism.

In the presence of glutamine, glucose, and 3-hydroxybutyrate, glutamine is the preferred oxidative substrate in enterocytes of suckling and weaned rats. Our studies of changes in intestinal metabolism in the developing rat clearly indicate that the oxidation of substrates that enter the citric acid cycle in the form of acetyl-CoA such as glucose, fatty acid, and lipids is low during the suckling period and increases after weaning. In contrast, glutamine that enters the citric acid cycle in the form of 2-oxoglutarate is high during the suckling period and does not change during weaning. The control of the citric acid cycle appears to be the intramitochondrial [NADH]/[NAD+] ratio, which is high during the suckling period and low in the intestine of weaned rats. These studies demonstrate how evaluating changes in metabolism during a natural perturbation such as weaning can identify control mechanisms of metabolism. Finally, the changes in substrate oxidation during weaning are not controlled by the endogenous steroid burst that occurs at 16 days of age. Substrate oxidation changes only after weaning, suggesting that a change in diet is a significant factor in intestinal substrate oxidation.

The effect of surgical bowel manipulation and anesthesia on intestinal glucose absorption in rats.

The effects of surgical bowel manipulation and anesthesia on intestinal glucose absorption were determined in chronically catheterized rats. Total and passive rates of glucose absorption were measured using 3-O-methyl-glucose (3OMG) and L-glucose, metabolically inert analogues of D-glucose. The rates of 3OMG absorption immediately postoperative and 4 h later were 86 and 62% less than the absorption rate 6 d postoperative. The absorption rates of 3OMG 1 and 2 d postoperative were not different from 6 d postoperative. Absorption of L-glucose was not altered by bowel manipulation and anesthesia. Even after correction for the increased resistance of the unstirred water layer (UWL) after bowel manipulation, the rates of total and active intestinal glucose absorption immediately postoperative were only 11 and 15% of predicted rates of absorption. In chronically catheterized rats, > 75% of luminal 3OMG at a concentration of 400 mM was absorbed by active transport. The Km and Vmax of 3OMG active transport corrected for the resistance of the UWL were 11.3 mM and 15.6 mumoles/min, respectively. We conclude that measurements of intestinal glucose absorption performed within 24 h of surgical bowel manipulation greatly underestimate active absorption even if corrections are made to account for the increased resistance of the UWL.

Active transport of 3-O-methyl-glucose by the small intestine in chronically catheterized rats.

A method is described for determining the fraction of intestinal 3-O-methyl-glucose (3OMG) absorption that occurs by active transport in chronically catheterized rats without the influence of anesthesia or surgical bowel manipulation. That fraction was determined by simultaneously measuring portal venous-aortic blood concentration gradients (delta C) of 3-O-methyl-glucose (3OMG) and L-glucose, metabolically inert analogues of D-glucose. 3OMG is actively and passively absorbed by the same mechanisms as D-glucose, L-glucose is only passively absorbed. The fraction of 3OMG that is actively transported was calculated from the difference between 3OMG and L-glucose absorption, divided by total 3OMG absorption. We found that more than 94% of 3-O-methyl-glucose is absorbed by active transport when luminal concentrations range from 50 to 400 mM. We conclude that in unrestrained, unanesthetized chronically catheterized rats, most 3OMG is actively absorbed by the intestine even at high luminal concentrations.

Where does it hurt? An interdisciplinary approach to improving the quality of pain assessment and management in the neonatal intensive care unit.

Identifying a premature infant's response to noxious stimuli and knowing when it is appropriate to intervene are major issues for the caregiver. After completing a pilot study, the staff of a 42-bed neonatal intensive care unit targeted improved pain assessment and management for further analysis. Using a series of Plan, Do, Check, and Act cycles they designed a stepwise set of interventions that resulted in the improved assessment and management of the neonate in pain.

Age-related changes in homeostasis of inorganic sulfate in male F-344 rats.

Advanced age is associated with a decline in renal function including decreased glomerular filtration rate, renal blood flow and renal tubular secretion. Endogenous inorganic sulfate homeostasis is maintained by concentration-dependent active renal reabsorption in the proximal tubule. The purpose of this study was to determine the effects of advanced age on: (1) the renal mechanisms for conserving endogenous inorganic sulfate and (2) the turnover of inorganic sulfate. Awake, male Fischer 344 rats age 4-5 months and 22-23 months received i.v. acetaminophen, 300 mg/kg, followed 2 h later by i.v. sodium sulfate, 2 mmol/kg, to lower and raise, respectively, plasma inorganic sulfate in order to measure the renal clearance of this anion from plasma at sub- and supraphysiologic concentration ranges. Another group of old and young male F-344 rats received a tracer injection of [35S]sodium sulfate to determine the effect of aging on the turnover of the endogenous inorganic sulfate pool. There was no statistically significant effect of advanced age on baseline plasma sulfate concentration or on the renal clearance of inorganic sulfate from plasma. The baseline excretion rate of inorganic sulfate in the senescent animals (0.38 +/- 0.25 mumol/min/kg, mean +/- S.D., n = 7) was significantly (P < 0.05) lower than that observed in the young animals (0.64 +/- 0.19 mumol/min/kg, n = 8). There was no difference in the turnover rate constant, as measured by the change in specific activity of urinary [35S]sodium sulfate, for the endogenous sulfate pool in old and young animals. Following acetaminophen administration, plasma sulfate concentrations declined similarly in young and old animals. Under the conditions of relative inorganic sulfate depletion, the renal excretion rate of inorganic sulfate decreased to zero in 7 of 8 young rats, whereas the old animals continued to excrete sulfate anion at an average rate of 23% of the baseline value. Aged animals have a defect in active tubular renal reabsorption of sulfate under conditions of sulfate depletion. Age-related changes in the total sulfate excretion rate may reflect changes in the metabolic fate of endogenous sulfate rather than changes in the endogenous production rate of this anion.

Changes in conjugative enzyme activity and acetaminophen metabolism in young and senescent male F-344 rats following prolonged exposure to buthionine sulfoximine.

This study examined how advanced age affects glucuronide and sulfate conjugation of acetaminophen after prolonged exposure to L-buthionine-S,R-sulfoximine (BSO) in male Fischer 344 rats. Young (4-5 month) and senescent (21-22 month) rats received 11 doses of BSO (2 mmol/kg) at 12-h intervals via a gastric cannula. Hepatic metabolism was assessed in vivo by measuring the products of reactions mainly responsible for acetaminophen elimination, namely the formation of the glucuronide and sulfate conjugates. Selected drug-metabolizing enzyme activities were also determined in vitro. BSO treatment increased the partial clearance to acetaminophen glucuronide by 90% and 41% in young and old rats, respectively, and similarly, induced p-nitrophenol and 1-naphthol UDP-glucuronosyl transferase activities to a greater extent in young versus senescent animals. Thus, the induction of these UDP-glucuronosyl transferase activities by BSO is preserved in senescent animals. Although the partial clearance to acetaminophen sulfate was decreased in senescent control rats compared to young controls, BSO treatment decreased the in vivo rate of sulfation in both age groups. Similar to previous findings with the Sprague-Dawley strain, BSO treatment did not induce hepatic cytochrome P-450 content or activity or cytosolic p-nitrophenol sulfotransferase activity in young and senescent Fischer 344 rats.

The oxidation of 3-hydroxybutyrate in developing rat jejunum.

Ketone bodies and glutamine are the primary oxidative substrates in the intestine of fasting adult rats. Because suckling rats consume the majority of their calories in the form of lipid and as a result have elevated blood ketone concentrations similar to fasting adult rats, we examined the role of ketone oxidation as a source of energy in the intestine of suckling rats. In suckling rats the rate of [3-14C]DL-3-hydroxybutyrate oxidation in intestinal tissue slices, enterocytes, and intestinal mitochondria was 0.15 +/- 0.01 nmol/h/mg wet wt, 8.6 +/- 1.1 nmol/h/mg protein and 8.3 +/- 1.3 nmol/h/mg protein, respectively. In suckling rats the rate of intestinal oxidation of [3-14C]DL-3-hydroxybutyrate was 75% lower than the rate in weaned rats in intestinal tissue slices, 55% lower in enterocytes, and 50% lower in intestinal mitochondria. The activity of two enzymes involved in ketone oxidation, 3-hydroxybutyrate dehydrogenase, and acetoacetyl-CoA thiolase was 38% and 55% higher in intestinal homogenates of suckling rats compared with adult rats. In contrast, 3-oxoacid CoA-transferase activity, an enzyme also involved in 3-hydroxybutyrate oxidation, was one third that of adults. These studies indicate that intestinal oxidation of 3-HB occurs in suckling rat pups, but is lower than the rate of oxidation in the intestine of weaned rats.