Improvement of the Mouse Grimace Scale set-up for implementing a semi-automated Mouse Grimace Scale scoring (Part 1).

The Mouse Grimace Scale (MGS) has been widely used for the noninvasive examination of distress/pain in mice. The aim of this study was to further improve its performance to generate repeatable, faster, blinded and reliable results for developing automated and standardized pictures for MGS scoring and simultaneous evaluation of up to four animals. Videos of seven C57BL/6N mice were generated in an experiment to assess pain and stress induced by repeated intraperitoneal injection of carbon tetrachloride (CCl4). MGS scores were taken 1 h before and after the injection. Videotaping was performed for 10 min in special observation boxes. For manual selection, pictures of each mouse were randomly chosen for quality analysis and scored according six quality selection criteria (0 = no, 1 = moderate, 2 = full accordance); the maximum possible score was 12. Overall, 609 pictures from six videos were evaluated for MGS scoring quality; evaluation was performed by using the picture selection tool or by manual scoring. With manual scoring, 288 pictures (48.3% of all randomly generated pictures) were deemed scorable using MGS (mean score = 22.15 ± SD 6.3). To evaluate the algorithm, ratings from different rater groups (beginner, medium-level trained, professional) were compared with the automated image generated. These differences were not significant (p = 0.1091). This study demonstrates an improved set-up and a picture selection tool that can generate repeatable, not-observer biased and standardized pictures for MGS scoring.

Effects of pneumoperitoneum on hemodynamic and systemic immunologic responses to peritonitis in pigs.

BACKGROUND: Experimental evidence supporting the safety of laparoscopic intervention during sepsis is limited. The purpose of this study was to evaluate the effects of pneumoperitoneum on immunologic and hemodynamic responses to peritoneal sepsis. MATERIALS AND METHODS: A porcine model of peritonitis was created using an intraperitoneal autologous fecal inoculum. Pigs were then subjected to one of four procedures 24 h postinoculation (n = 6 per group): laparotomy, CO(2) laparoscopy, helium laparoscopy, and anesthesia only (1.5% isoflurane in 100% O(2), mechanical ventilation). Venous blood samples were obtained prior to inoculation, and at 24 (prior to procedure), 30, 48, 72, and 96 h postinoculation to determine white blood count (WBC) with differential, C-reactive protein (CRP), tumor necrosis factor, and bacteremia. Heart rate, end-tidal CO(2) (ETCO(2)), mean arterial blood pressure (MAP), and arterial blood gas variables were also measured at baseline and every 30 min throughout the procedure. RESULTS: Postoperative blood cultures confirmed systemic bacteremia in all groups at all time periods postinoculation. Following inoculation, WBC, band cell count, and CRP remained elevated above baseline in all groups throughout the study (P < 0.01). However, no significant differences in these parameters were observed among groups. In the CO(2) laparoscopy group, MAP, ETCO(2), and arterial pCO(2) were increased above baseline, while pH was decreased throughout the procedure (P < 0.01). CONCLUSIONS: In this animal model of peritonitis, CO(2) pneumoperitoneum induced hypercapnia, acidemia, and systemic hypertension intraoperatively, without a discernable effect on systemic immune function.

Histologic evaluation of fetal brains following maternal pneumoperitoneum.

BACKGROUND: The purpose of this study was to determine if maternal pneumoperitoneum with carbon dioxide (CO2) produces evidence of central nervous system (CNS) injury in preterm fetal guinea pigs. METHODS: Thirty pregnant guinea pigs at gestational day (GD) 45 were assigned at random to one of three treatment groups: anesthesia only, CO2 pneumoperitoneum (5 mmHg), or laparotomy. Dams were killed 3 or 5 days postprocedure and fetal brains (83 total) harvested and fixed for subsequent histopathologic evaluation. For comparative purposes, histologic features of fetal guinea pig brain injury were defined from examination of fetal brains harvested from an additional dam that underwent laparotomy with 20 min of uterine arterial occlusion. RESULTS: Carbon dioxide pneumoperitoneum did not increase maternal/fetal morbidity. No evidence of brain injury was found in fetuses from any of the treatment groups. CONCLUSION: Carbon dioxide pneumoperitoneum at 5 mmHg for 40 min in the pregnant guinea pig does not produce evidence of fetal brain injury.

The management of nonpigmented gallstones in children.

BACKGROUND/PURPOSE: An increasing number of children are diagnosed with gallstones today. The best management of nonpigmented gallstones in children without hematologic disorders is not known. METHODS: The authors prospectively studied 74 children with cholelithiasis diagnosed with ultrasonography. Clinical presentation, natural history, complications, and indications for cholecystectomy were examined. The follow-up (mean, 21 months) consisted of routine clinic visits, chart reviews, and telephone questionnaires with the children or their parents. RESULTS: Of the 74 children, 33 required cholecystectomies, and 41 were followed. The average age was similar in the 2 groups (11.7 v 11.0 years). Children with risk factors for cholelithiasis required earlier surgical treatment (P < .001). In the operative group, 8 presented acutely and 25 electively. There were 2 complications, a wound infection and a retained common duct stone. In the group that underwent follow-up, 34 of 41 children remained asymptomatic or had symptoms improve with dietary manipulation. No complications developed during the follow-up period. CONCLUSIONS: Children with gallstones and typical symptoms of right upper quadrant or epigastric pain with food intolerance should undergo cholecystectomy. Eighty-two percent of children with cholelithiasis and atypical symptoms had improvement with dietary manipulation. Pediatric patients with gallstones that are asymptomatic or associated with atypical symptoms can be safely followed without complications.

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes.

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

Prenatal therapy for pericardial teratomas.

Although the majority of reported pericardial teratomas have been excised successfully in the postnatal period, the outcome of prenatally diagnosed pericardial teratomas remains less favorable. Two recent cases of prenatally diagnosed pericardial teratomas and a review of previously reported cases suggest a new management algorithm for those pericardial teratomas discovered in utero. Fetuses in whom hydrops does not develop may be safely followed up and treated postnatally. However, if hydrops develops, the fetus requires treatment with either aspiration of the pericardial effusion, or fetal surgery and resection.

Gastroschisis increases small bowel nitric oxide synthase activity.

In gastroschisis, the eviscerated fetal bowel frequently is damaged and this results in hypoperistalsis and malabsorption. The mechanistic link that ties gastroschisis-induced intestinal damage to dysfunction may be nitric oxide (NO) and the enzyme responsible for producing it, NO synthase. Using a fetal rabbit model, the authors investigated the hypothesis that the hypoperistalsis and malabsorption associated with gastroschisis may be attributable to abnormal small bowel NO synthase activity. Using the 3H-arginine-to-3H-citrulline conversion assay, they measured NO synthase activity in the small bowel of full-term fetal rabbits with and without gastroschisis. The mean total small bowel NO synthase activity of fetal rabbits with gastroschisis was 2.5 times greater than that of control littermates without gastroschisis (n = 6; 5,726 +/- 834 v 2,208 +/- 537 mean pmol/mg protein/min; P = .004). This increased NO synthase activity also was studied by measuring the individual isoforms of NO synthase, and the site of increased NO synthase activity was localized to the small bowel epithelium and neurons. After detecting and localizing the gastroschisis-induced increase in NO synthase activity, the authors explored the mechanism of this increase using NADPH-diaphorase staining. With this histological staining technique, no quantitative increase was found in the small bowel NO synthase of the rabbits with gastroschisis. This suggests that the increased NO synthase activity found in these rabbits is the result of accelerated enzyme kinetics. These findings suggest that the increased NO synthase activity caused by gastroschisis may contribute to the common clinical sequelae of malabsorption and intestinal dysmotility.

Fetal endoscopic ('Fetendo') tracheal clip.

Fetal surgery can correct several life-threatening malformations before birth. Despite recent advances in fetal surgery, preterm labor remains a major problem directly related to the large uterine incision required for fetal exposure. Fetal endoscopic surgery ("Fetendo") obviates the need for a large uterine incision and may reduce the overall risks of fetal surgery by causing less uterine trauma and ultimately less preterm labor. Temporary tracheal occlusion is a promising strategy to enlarge the lungs in fetuses with congenital diaphragmatic hernia. Using the technology developed for laparoscopic surgery and for temporary tracheal occlusion, we have developed an endoscopic technique for tracheal occlusion with an endoscopic clip in a fetal sheep model. The evolution of this technique may allow temporary tracheal occlusion without incisional hysterotomy or maternal laparotomy.

Immediate reconstruction for penile agenesis.

The goal of treatment for penile agenesis is early female gender assignment and feminizing reconstruction of the perineum. Historically, this required multiple operations including both vesicostomy and colostomy. The present case demonstrates the feasibility of early total reconstruction through a posterior sagittal approach that avoids diversion of stool and urine.

Transesophageal echocardiography in fetal sheep. A monitoring tool for open and fetoscopic cardiac procedures.

BACKGROUND: Cardiac procedures in exteriorized fetuses or assisted by fetoscopy require monitoring capabilities not attended by conventional maternal transabdominal echocardiography. METHODS: We, therefore, assessed the potential of fetal transesophageal echocardiography (TEE) utilizing an intravascular ultrasound catheter (IVUC) for fetal cardiac monitoring. We inserted a 10-F-10-MHz IVUC into the esophagus in 12 exteriorized fetal sheep and by a fetoscopic approach in 4 fetal sheep. Cardiac events were observed. Heart rate, cardiac rhythm, patency of the foramen ovale and ductus arteriosus, and the width of the branch pulmonary arteries could be assessed in all fetuses. Ventricular contractility could be assessed only in fetuses weighing less than 2.5 kg. Larger fetuses did not allow adequate imaging of the apical portion of the ventricles because of limited tissue penetration of the IVUC. Fetal TEE permitted placing small guide wires in the cardiac atria and left ventricle. Short-lived premature beats following intracardiac manipulations of these wires could be observed by fetal TEE in all cases. RESULTS: At autopsy, no complications from IVUC insertion were observed in the exteriorized fetuses. Fetoscopic placement of the IVUC resulted in minor perioral skin erosion in two nonexteriorized fetuses. CONCLUSIONS: In conclusion, fetal TEE can be achieved with minor fetal injury and may provide useful information during open and fetoscopic cardiac procedures. Further improvements in IVUC design will permit the application of this technique to monitor human fetal cardiac procedures.

Challenging the embryogenesis of cloacal exstrophy.

As presently understood, cloacal exstrophy results from a migration failure of the lateral mesodermal folds of the infraumbilical anterior abdominal wall, and rupture of the resulting enlarged, persistent cloacal membrane before the eighth week of gestation. The authors present ultrasonographic evidence that disputes this embryological theory. Routine ultrasonography of a twin gestation at 18 weeks showed that one twin had a dilated cloacal abnormality, bilateral hydronephrosis, and oligohydramnios. Repeat ultrasonography at 24 weeks demonstrated rupture of the cloacal anomaly, with resolution of both the hydronephrosis and oligohydramnios. This twin was born with classic cloacal exstrophy. This striking ultrasound evidence of an intact cloacal membrane at 18 weeks, which ruptured before 24 weeks, relieving the urinary tract outlet obstruction, forces us to rethink how this surgically correctable anomaly develops.

Percutaneous access to the uterus for fetal surgery.

In utero repair of selected life-threatening malformations in the human fetus is now a clinical reality, yet fetal surgery continues to pose significant risks to both the mother and the unborn child. Preterm labor is a major problem directly related to the large uterine incision required for fetal exposure. Using technology developed for laparoscopic surgery, we have devised instruments and techniques to perform fetal endoscopic surgery. We now report a percutaneous technique for direct endoscopic access to the uterus. Minimally invasive fetoscopic surgery may expand the indications for fetal surgery by decreasing fetal risks, facilitating intervention earlier in gestation, and reducing preterm labor. This technique was developed in 4 fetal lambs who underwent endoscopic intervention at 105-110 days gestation (term = 145 days). Under ultrasound guidance, a 20-gauge spinal needle was advanced through the maternal abdomen, uterus, and directly into the amniotic cavity. Warmed saline was infused through the needle to expand the amniotic cavity. Next, a 5-mm balloon-tipped trocar was placed percutaneously with ultrasound guidance into the amniotic cavity. A 5-mm laparoscope was introduced and under endoamniotic vision two more 5-mm trocars were percutaneously placed. In all four sheep a 5-mm trocar was placed percutaneously into the gravid uterus. The most difficult step was puncturing through the amniotic membranes, but the sharp tip of the trocar facilitated getting into the amniotic cavity. Excellent visualization of the fetus was obtained with minimal uterine trauma. We have developed a fetoscopic technique in sheep for percutaneous placement of trocars into the uterus using ultrasound guidance. This approach allowed excellent visualization of the fetus with significantly less uterine trauma than open fetal surgery and is an essential prerequisite for future fetal endoscopic interventions.

Using a research-based assessment scale in clinical practice.

The Braden Scale has been translated into Chinese, Japanese, Dutch, French, German, Italian, and perhaps other languages. It has been implemented in many health care settings in the United States and abroad. Perhaps the most significant data come from individual settings, such as the two highlighted earlier, which show that through use of a research-based risk assessment tool and a program of prevention based on the findings of the assessment, pressure ulcer incidence can be reduced. Both sites saw a 50% to 60% decrease in incidence of pressure sores. If the cost projections of Miller and Delozier cited earlier are correct and this decrease could be replicated in hospitals across the country, the cost savings for the nation would exceed $400 million.

Correlation of in vitro fluconazole resistance of Candida isolates in relation to therapy and symptoms of individuals seropositive for human immunodeficiency virus type 1.

Yeast strains isolated from the oropharynx of 87 consecutive patients infected with human immunodeficiency virus type 1 were examined for in vitro susceptibility to fluconazole. Candida albicans was isolated from 73 patients. Fifty-one patients had received antifungal therapy in the month preceding the yeast infection. Thirty-two patients had symptomatic oropharyngeal candidiasis. The MICs were correlated with azole use and with clinical symptoms and signs. Although there is overlap between groups, in vitro testing identified a large group of patients for whose yeast isolates the fluconazole MICs were high and who remained symptomatic while receiving azole therapy. This study supports the ability of in vitro testing to predict the clinical outcome of mucosal fungal infections. The study also demonstrates that azole resistance of oropharyngeal yeasts is a common problem in patients infected with human immunodeficiency virus type 1 and that this azole resistance has clinical relevance.

Serotonin 5-HT2 receptor binding on blood platelets as a state dependent marker in major affective disorder.

Serotonin receptors of the 5-HT2 type were studied on platelet membranes from 15 patients suffering from major depression. Receptor binding and clinical state (assessed by the Hamilton and Beck rating scales) were examined in a drug free state upon admission and after 1 and 3 weeks of treatment with the antidepressant maprotiline (MPT). 5-HT2 receptor binding changed in correlation with changes in the clinical state of the patients as judged by the rating scales. Since most patients showed a clinical improvement, the patients as a group exhibited a significant decrease in binding concomitant with a drop in depression scores. However, in those patients in whom there was no clinical change or an increase in depression scores, 5-HT2 receptor binding did not change or increased, respectively, thus resulting in a significant correlation between clinical changes and changes in binding. These results support the use of 5-HT2 receptors on platelets in evaluating depression and its treatment.