Staged surgical approach of neonates with esophageal atresia and tracheoesophageal fistula from low- and middle-income countries.

BACKGROUND: Neonates born in low- and middle-income countries (LMICs) with esophageal atresia (EA) and tracheoesophageal fistula (TEF) often do not have access to adequate surgical care. We have partnered with the non-profit organization World Pediatric Project (WPP) to facilitate care for such patients. METHODS: Our protocol included placement of a gastrostomy tube by local surgeons before definitive repair at the Children's Hospital of Richmond at VCU (CHoR). The outcomes of WPP-sponsored patients with EA and TEF in this program (n = 9) were compared to patients born in the U.S. with the same condition at CHoR (n = 9). RESULTS: The baseline characteristics of the groups aside from the age at admission to CHoR and at day of surgery were comparable (0.5 ± 1.3 days vs. 15.3 ± 11.1 days, p < 0.0001; 1.1 ± 3.9 days vs. 37.3 ± 8.3 days, p < 0.0001, respectively). All patients received definitive repair without mortality; the average length of stay after surgery was lower in the WPP group. There were 4 surgical complications in the U.S. group and 1 in WPP group. All patients were weaned off of TPN and all WPP patients are now on a regular PO diet. Every WPP patient has been seen in follow-up in the U.S. and in their home country. CONCLUSION: We provided successful multidisciplinary care for neonates with EA and TEF from LMICs with outcomes similar to the neonates with the same abnormality in the U.S. The partnership with WPP has been invaluable as it has provided essential support to identify and manage these patients. LEVEL OF EVIDENCE: IV.

Providing care to children from low and middle-income countries with complex surgical problems: An 18 year review.

PURPOSE: The burden of surgical disease in children from low and middle-income countries (LMICs) is becoming more recognized as significant and undertreated.  We recently reviewed our health system's experience with providing quaternary-level surgical care to children from LMICs through a partnership with World Pediatric Project (WPP), a not-for-profit organization. METHODS: A retrospective review was performed of all WPP-sponsored patients who received surgical care at our children's hospital from LMICs in the Caribbean and Central America from July 2000 to August 2018. RESULTS: Two hundred and fifty-five patients (average age: 5.9 ± 5.3 years; range: <1-18 years) from 14 countries received a total of 371 moderately to significantly complex operations from 10 pediatric surgical subspecialties, with cardiac, neurosurgery, craniofacial and general/thoracic surgical subspecialties being the most common. The average length of hospital stay was 10.7 ± 18.9 days.  All patients had the opportunity to follow-up with local providers and/or visiting WPP-sponsored surgical teams. 227 patients (93.8%) were seen by WPP providers or released to an in-country physician partnering with WPP. There were 21 (8.2%) total, minor and major, postoperative complications.  Five deaths (2.0%) occurred at our institution and 7 from disease progression, after returning to their home country. CONCLUSIONS: Providing complex surgical care to LMIC children in the US may help address a significant global burden.  This care can be provided by multiple subspecialists with excellent outcomes, good follow-up, and low complication and mortality rates.  Having a supportive health care system, volunteer surgeons, and an organization that manages logistics and provides financial support is essential. TYPE OF STUDY: Clinical research, retrospective review LEVEL OF EVIDENCE: Level IV.

Novel and emerging devices and operations in the treatment of obesity in children and adolescents.

Obesity is among the most common and costly chronic disorders worldwide. Estimates suggest that in the United States obesity affects one-third of adults, accounts for up to one-third of total mortality, is concentrated among lower income groups, and increasingly affects children as well as adults. A lack of effective options for long-term weight reduction magnifies the enormity of this problem; individuals who successfully complete behavioral and dietary weight-loss programs eventually regain most of the lost weight. We included evidence from basic science, clinical, and epidemiological literature to assess current knowledge regarding mechanisms underlying excess body-fat accumulation, the biological defense of excess fat mass, and the tendency for lost weight to be regained. A major area of emphasis is the science of energy homeostasis, the biological process that maintains weight stability by actively matching energy intake to energy expenditure over time. Growing evidence suggests that obesity is a disorder of the energy homeostasis system, rather than simply arising from the passive accumulation of excess weight. We need to elucidate the mechanisms underlying this "upward setting" or "resetting" of the defended level of body-fat mass, whether inherited or acquired. The ongoing study of how genetic, developmental, and environmental forces affect the energy homeostasis system will help us better understand these mechanisms and are therefore a major focus of this statement. The scientific goal is to elucidate obesity pathogenesis so as to better inform treatment, public policy, advocacy, and awareness of obesity in ways that ultimately diminish its public health and economic consequences.

A pilot study of laparoscopic gastric plication in adolescent patients with severe obesity.

BACKGROUND: Metabolic and bariatric surgery in adolescents with severe obesity has been demonstrated to be safe and have long-term treatment efficacy. However, less than 0.1% of adolescents meeting criteria undergo surgery. METHODS: A prospective pilot study of adolescents who underwent laparoscopic gastric plication (LGP) was conducted to evaluate safety and effects on anthropometrics and weight-related medical and psychological comorbidities. RESULTS: Four adolescents enrolled and underwent LGP; two withdrew 90 days postoperatively and two were followed through 36 months. Preprocedure body mass index was 41.7-53.7 kg/m2 with decreases in % change of BMI of 17.5% and 39.7% at 36 months after surgery. Patients reported early satiety, good hunger control, and limited nausea at all time points. There were no major complications. Mild gastroesophageal reflux and abdominal pain occurred; both resolved without surgical intervention. Minor improvements in psychological comorbidities were also reported. CONCLUSIONS: LGP can be safely performed in adolescents with severe obesity and achieves modest weight loss. Although limited by its small sample size, this study provides preliminary support for LGP as an appropriate surgical option for adolescents. A larger, multidisciplinary study is planned. TYPE OF STUDY: Prospective case series review. LEVEL OF EVIDENCE: Level IV evidence: case series without comparison.

Utilization of a handheld telemedicine device in postoperative pediatric surgical care.

PURPOSE: The purpose of this study was to assess the utilization of a handheld telemedicine (TM) device in the postoperative care of pediatric surgical patients. METHODS: We performed postoperative TM evaluations using an advanced medical tablet immediately prior to seeing the patients in clinic as well as at two different time points from their home. The caregivers and physicians were surveyed about their overall satisfaction. RESULTS: Twenty-four postoperative patients who underwent a variety of general surgical operations were included. There were no changes to the TM plan of care following "in person" evaluations (n = 12) and no complications, missed diagnoses, emergency department visits, or additional clinic visits in those who only had TM postoperative evaluations (n = 12). Caregiver satisfaction ratings were 3.92 ±â€¯0.28 out of 4 (4 = very satisfied). Ninety-two percent of caregivers responded that they would be comfortable with a TM-only postoperative evaluation in the future. The physician was able to formulate an accurate assessment and plan using the device. The average travel distance saved was 44.7 ±â€¯45.5 miles (range = 10-150 miles). CONCLUSIONS: These preliminary data suggest safe and effective care with high caregiver and physician satisfaction can be provided by utilizing TM in the postoperative care of pediatric surgical patients. LEVEL OF EVIDENCE: IV.

Minimally Invasive Pediatric Surgery During Remote Humanitarian Missions Is Feasible, Safe, and Effective.

PURPOSE: There remains a critical need for the provision for pediatric humanitarian aid worldwide. Historically, the emphasis of global pediatric health needs has been focused on infectious diseases. Today, we are witnessing a shift in this paradigm, with growing attention being paid toward the surgical needs of children. The use and deployment of minimally invasive surgery (MIS) in these austere environments with its concomitant reduction in length of hospitalization, pain, and morbidity is logical. The goal of this study was to report our deployment strategy and review our experience with pediatric MIS during humanitarian missions to determine if it is safe, feasible, and efficacious. METHODS: As part of the World Pediatric Project (WPP), data were collected retrospectively from the general pediatric surgery (GPS) team missions from January 2007 to January 2017. All cases were performed at a single medical center in the Eastern Caribbean Island Nation of St. Vincent and the Grenadines (SVG). Data included patient demographics, diagnosis, procedure, conversion to open procedure, complications, and postoperative course. The teams utilized a dedicated WPP operating theater, prepositioned and deployed GPS supplies, and MIS resources. All anesthesia, surgical, and nursing personal were board certified and trained professionals functioning as part of the WPP team. RESULTS: One hundred thirty-four children underwent general and thoracic pediatric surgical procedures during the study period. Mean age 9.2 years (2-19 years). Thirty-seven children underwent MIS procedures (27%). There were no conversions to open procedures. There were only two postoperative complications, cellulitis following laparoscopic appendicostomies, which required intravenous antibiotics and were discharged on a course of oral antibiotics. The postoperative course for all children was uneventful and no child required readmission. There were no technical failures in the MIS systems or instrumentation. CONCLUSIONS: Our retrospective review supports the use of MIS techniques as part of GPS humanitarian missions. We have found it to be a safe, feasible, and effective modality that may reduce length of stay, pain, and morbidity compared with open procedures in these remote environments. Although our MIS systems and instrumentations functioned effectively, concerns regarding the storage and sustainability for future missions are significant. Onsite health care partners, redundant systems, and remote technical support access could potentially alleviate these concerns.

Laparoscopic gastroesophageal dissociation in neurologically impaired children with gastroesophageal reflux disease.

PURPOSE: Neurologically impaired children with severe gastroesophageal reflux disease (GERD) are a challenging group of patients. We theorized that a laparoscopic gastroesophageal dissociation (LGED) may decrease reflux-related readmissions and healthcare visits, and improve quality of life (QOL) for them and their caregivers. METHODS: A retrospective review was performed on our pediatric patients that underwent an LGED along with a caregiver survey from 2013 to 2017. RESULTS: Twenty-two neurologically impaired patients (14months-17years) with severe GERD underwent an LGED. Patients weighed 7.9-57kg (avg=23.8kg), length of stay ranged from 5 to 20days (avg=12days), estimated blood loss ranged from <5cm3 to 450cm3 (avg=66cm3, median=25cm3), and duration of operation ranged from 299 to 641min (avg=462min). One death occurred on postoperative day 19 from gram negative sepsis (30-day perioperative mortality of 4.5%). There were a modest number of minor and major complications (follow-up avg.=13.7months, range=2-40months). There was a decrease in healthcare visits for respiratory illnesses (rated 5/5 from all 13/19 survey respondents) as well as improvements in perceived QOL of the patient (avg=4.3/5) and caregiver (avg=4.6/5). CONCLUSIONS: Our cohort of patients had a reduction in readmissions and healthcare visits, and improved QOL after undergoing an LGED based on the perceptions of their caregivers. In neurologically impaired patients with severe GERD, an LGED may be a viable alternative to traditional treatments. TYPE OF STUDY: Retrospective case series review. LEVEL OF EVIDENCE: Level IV evidence: case series without comparison.

A treatment program for babies with esophageal atresia in Belize.

BACKGROUND: Children born with congenital anomalies in low- and middle-income countries often face a multitude of challenges. Access to pediatric surgical services is limited because of a lack of medical facilities, an adequate transportation system, and a lack of trained surgeons, anesthesiologists, and nurses, all of which leads to a high mortality rate. METHODS: This is a report of a 5-y collaborative effort between the World Pediatric Project, the Children's Hospital of Richmond at Virginia Commonwealth University, and multiple organizations within the country of Belize to provide care for infants born with esophageal atresia, with or without associated tracheoesophageal fistula. RESULTS: A total of six infants were transferred to our institution in Richmond, VA for operative correction of their esophageal atresia. CONCLUSIONS: Caring for infants with congenital anomalies can be challenging, especially children from low- and middle-income countries. Through collaboration between countries and nonprofit organizations, life-saving international care can be provided to children for these conditions.

Histrelin for central precocious puberty-a single surgeon experience.

BACKGROUND: The most common cause of precocious puberty is idiopathic central precocious puberty (CPP), which is usually treated with monthly injections. An alternative treatment of precious puberty is a subcutaneous implant that contains histrelin acetate, which is continuously released for more than 1 y and then removed or replaced with a new implant. METHODS: The aim of this study was to conduct a retrospective review of one surgeon's experience with the histrelin implant and to examine patient satisfaction. After obtaining institutional review board approval, the charts of all children who had undergone at least one implant were reviewed. RESULTS: Fifty-eight children, average age 8.4 y old (range 7-14), underwent at least one histrelin implant insertion for treatment of CPP. Parents of 44 patients were able to be reached by telephone for this study and rated the histrelin implant treatment highly. All implants were placed, replaced, or removed without significant difficulty, and there were no complications. CONCLUSIONS: This study suggests that the use of a histrelin subcutaneous implant for control of CPP is a safe and effective method for the treatment of this condition.

Laparoscopic gastric plication in the morbidly obese adolescent patient.

Childhood obesity is a significant problem. Due in part to suboptimal weight loss with lifestyle intervention alone, bariatric surgery, combined with ongoing lifestyle changes, has become a favorable approach in adolescents with severe obesity and weight-related comorbidities and is associated with effective weight loss and reducing weight-related comorbidities. Laparoscopic greater curvature plication is a promising new bariatric surgical procedure that has been shown to be effective in adults with severe obesity but has not been evaluated in the adolescent population. Gastric plication may be a particularly attractive approach for the adolescent patient as it is potentially reversible, does not involve the surgical removal of tissue, and is without a significant malabsorptive component. Our team has obtained approval from our Institutional Review Board to perform a laparoscopic greater curvature plication on 30 adolescent patients with severe obesity and study its effect on weight loss, metabolic effects, and psychological functioning in the setting of a multidisciplinary program. Results of this study, including comprehensive clinical and psychological data collected over a 3.5-year span, will inform larger prospective investigations comparing the laparoscopic greater curvature plication and other bariatric operations in the adolescent population.

Pediatric health care provider perceptions of weight loss surgery in adolescents.

This study explored pediatric health care providers' obesity treatment practices and perceptions about adolescent weight loss surgery (WLS). Surveys were e-mailed to pediatric listservs. After descriptive analyses, correlations, chi-squares, and one-way analyses of variance compared responses by provider characteristics. Surveys were completed by 109 providers. Almost half do not routinely measure body mass index. Providers typically counsel patients about lifestyle change, with limited perceived benefit; <10% have ever referred patients for WLS, citing cost (20%), risk (49%), or "not indicated in pediatrics" (17%) as reasons. However, when presented with patient scenarios of different ages and comorbidities, likeliness to refer for WLS increased substantially. Surgeons, younger providers and those with fewer years of experience were more likely to refer for WLS (P < .05). Despite expert consensus recommendations supporting WLS as part of a comprehensive obesity treatment plan, significant pediatric provider resistance to refer obese adolescents remains. Improved referral and management practices are needed.

The effects of irrigation on outcomes in cases of perforated appendicitis in children.

INTRODUCTION: Appendicitis is the most common indication for urgent abdominal operation in children. Approximately 20%-30% of patients will have a perforation at operation. Intra-abdominal abscess after appendectomy is reported in 3%-20% of patients and adds significantly to hospital stay with increased morbidity and overall cost. Surgical dogma has long advocated for irrigation in the setting of gross pus to prevent abscess formation. METHODS: Following IRB approval, data were retrospectively collected for children who had undergone appendectomy for perforated appendicitis at one of two children's hospitals over the course of 5 y. Perforation was determined by review of operative notes. All patients had free fluid in their peritoneal cavity evacuated by suction, whereas some of the patients also had their peritoneal cavity irrigated with normal saline. Postoperative intra-abdominal abscess rates were determined based on clinical symptoms and confirmatory radiologic studies. RESULTS: There were 99 patients in the suction-only group and 139 in the irrigation group. Standard demographics were relatively similar between the two groups. There were significantly lower rates of intra-abdominal abscess formation (4.0% versus 17.2%, P = 0.002) and wound infection (1.0% versus 8.6%, P = 0.003) in the suction-only group compared with the irrigation group. We further analyzed abscess rates by surgical treatment, either laparoscopic or open appendectomy. There were 85 patients in the laparoscopic group and 152 patients in the open appendectomy group. In this subgroup analysis, there were also significantly lower rates of abscess formation in patients treated with suction only compared with irrigation in the laparoscopic (3.5% versus 18.8%, P = 0.012) and open appendectomy groups (4.2% versus 16.3%, P = 0.036). CONCLUSIONS: Results of this retrospective review indicate that a suction-only approach significantly decreased rates of abscess formation and wound infections compared to irrigation in cases of perforated appendicitis in children.

Irrigation versus suction alone during laparoscopic appendectomy for perforated appendicitis: a prospective randomized trial.

BACKGROUND: The efficacy of irrigating the peritoneal cavity during appendectomy for perforated appendicitis has been debated extensively. To date, prospective comparative data are lacking. Therefore, we conducted a prospective, randomized trial comparing peritoneal irrigation to suction alone during laparoscopic appendectomy in children. METHODS: Children younger than 18 years with perforated appendicitis were randomized to peritoneal irrigation with a minimum of 500 mL normal saline, or suction only during laparoscopic appendectomy. Perforation was defined as a hole in the appendix or fecalith in the abdomen. The primary outcome variable was postoperative abscess. Using a power of 0.8 and alpha of 0.05, a sample size of 220 patients was calculated. A battery-powered laparoscopic suction/irrigator was used in all cases. Pre- and postoperative management was controlled. Data were analyzed on an intention-to-treat basis. RESULTS: A total of 220 patients were enrolled between December 2008 and July 2011. There were no differences in patient characteristics at presentation. There was no difference in abscess rate, which was 19.1% with suction only and 18.3% with irrigation (P = 1.0). Duration of hospitalization was 5.5 ± 3.0 with suction only and 5.4 ± 2.7 days with group (P = 0.93). Mean hospital charges was $48.1K in both groups (P = 0.97). Mean operative time was 38.7 ± 14.9 minutes with suction only and 42.8 ± 16.7 minutes with irrigation (P = 0.056). Irrigation was felt to be necessary in one case (0.9%) randomized to suction only. In the patients who developed an abscess, there was no difference in duration of hospitalization, days of intravenous antibiotics, duration of home health care, or abscess-related charges. CONCLUSIONS: There is no advantage to irrigation of the peritoneal cavity over suction alone during laparoscopic appendectomy for perforated appendicitis. The study was registered with clinicaltrials.gov at the inception of enrollment (NCT00981136).

Robot-assisted thoracoscopic thymectomy for treating myasthenia gravis in children.

INTRODUCTION: In the United States, the prevalence of myasthenia gravis (MG) is approximately 14-20 per 100,000. One treatment option involves a thymectomy, which can lead to remission of symptoms. The amount of thymic tissue removed is correlated with a better outcome for patients. Thus, it is critical that the procedure used when performing a thymectomy maximize the resection of thymic tissue. Robotic-assisted thoracoscopic thymectomy provides a minimally invasive platform that avoids the mortality and morbidity of a median sternotomy while providing better visualization and a more delicate dissection than is available in a standard thoracoscopic procedure. PATIENTS AND METHODS: Following Institutional Review Board approval, in total, 9 patients who underwent robotic thymectomy were reviewed. Intraoperative statistics such as operative time and blood loss were reviewed from operative records. Postoperative outcomes such as hospital stay, discharge medications, and complications were reviewed from hospital charts. Lastly, disease response was evaluated in consultation with a pediatric neurologist who specializes in MG. RESULTS: Age at operation ranged from 2 to 15 years of age (average, 9.4 years). A majority of patients had an MGFA classification of II or greater (n=5). All patients were on pyridostigmine preoperatively, and 7 of 9 (77%) were taking prednisone. Mean operative time was 160.1±6.1 minutes. Average postoperative hospital stay was 1.1±0.3 days. One patient had a documented persistent pneumothorax on postoperative Day 1, which was treated with nasal cannula oxygen for an additional day. There were no additional operative complications, and all patients were discharged home on acetaminophen with codeine for pain control. Eight of 9 patients had improvement in MG symptoms after the procedure. CONCLUSIONS: Robotic-assisted thoracoscopic thymectomy is a safe and effective operation for children with MG. Robotic assistance allows for articulating instruments, three-dimensional visualization, and minimal blood loss. These factors may allow for a more complete resection compared with a standard thoracoscopic thymectomy.

Robotic-assisted laparoscopic Nissen fundoplication with gastrostomy preservation in neurologically impaired children.

Robotic fundoplication has equivalent safety profiles, hospital stay, and time to alimentation, compared to laparoscopic fundoplication, but is not indicated for routine repair due to higher cost, decreased availability, and longer procedure time. Robotic surgery does offer key advantages over standard laparoscopy by employing internally articulating arms, a stable camera platform, and three dimensional imaging. Children presenting for initial or redo fundoplication after feeding gastrostomy are a subset of patients that may benefit from the robotic approach. Minimal dissection of the phrenoesophageal ligament, in combination with four anchoring sutures from the esophagus to the crura, has been shown to lead to less wrap herniation in children. This technique is particularly difficult in standard laparoscopy without dislodgement of the gastrostomy, particularly if there are abundant adhesions or a replaced left hepatic artery to preserve. In this article, we present 15 children with neurologic impairment and previous gastrostomy who underwent Nissen fundoplication, using the da Vinci surgical robot (Intuitive Surgical, Inc., Sunnyvale, CA). All patients underwent a floppy Nissen fundoplication after crural closure and placement of four anchoring stitches to the crura. Six patients (40%) had redo Nissens and 5 (33.3%) had replaced left hepatic or accessory arteries that were preserved. Seven patients underwent repair of a hiatal hernia and 2 had biologic mesh placed. There were no conversions to open or intraoperative complications. One child had a revision of the gastrostomy site, because the prior percutaneous endoscopic gastrostomy had been placed through the transverse mesocolon. There were only a few minor postoperative complications. All children were doing well at latest follow-up (average, 32 months). The da Vinci surgical robot can be used to safely perform fundoplications in patients with gastrostomy tubes. The articulating instruments allow for the optimal placement of four crural tacking sutures, while preserving the gastrostomy, even in the presence of a replaced left hepatic artery.

Clinical clearance of the cervical spine in blunt trauma patients younger than 3 years: a multi-center study of the american association for the surgery of trauma.

BACKGROUND: Cervical spine clearance in the very young child is challenging. Radiographic imaging to diagnose cervical spine injuries (CSI) even in the absence of clinical findings is common, raising concerns about radiation exposure and imaging-related complications. We examined whether simple clinical criteria can be used to safely rule out CSI in patients younger than 3 years. METHODS: The trauma registries from 22 level I or II trauma centers were reviewed for the 10-year period (January 1995 to January 2005). Blunt trauma patients younger than 3 years were identified. The measured outcome was CSI. Independent predictors of CSI were identified by univariate and multivariate analysis. A weighted score was calculated by assigning 1, 2, or 3 points to each independent predictor according to its magnitude of effect. The score was established on two thirds of the population and validated using the remaining one third. RESULTS: Of 12,537 patients younger than 3 years, CSI was identified in 83 patients (0.66%), eight had spinal cord injury. Four independent predictors of CSI were identified: Glasgow Coma Score <14, GCSEYE = 1, motor vehicle crash, and age 2 years or older. A score of <2 had a negative predictive value of 99.93% in ruling out CSI. A total of 8,707 patients (69.5% of all patients) had a score of <2 and were eligible for cervical spine clearance without imaging. There were no missed CSI in this study. CONCLUSIONS: CSI in patients younger than 3 years is uncommon. Four simple clinical predictors can be used in conjunction to the physical examination to substantially reduce the use of radiographic imaging in this patient population.

Scarless fetal mouse wound healing may initiate apoptosis through caspase 7 and cleavage of PARP.

INTRODUCTION: Apoptotic mechanisms are thought to be important in wound healing for the removal of inflammatory cells and evolution of granulation tissue. However, little is understood about the signal, propagation, and mechanisms responsible for triggering cell death in tissue injury, particularly during fetal wound repair. Understanding these signals may lead to insights regarding scarless wound healing. We hypothesized that differences in apoptosis would exist in mid- (E15) compared with late-gestational (E18) mice subjected to cutaneous wounds. We examined early apoptotic signals that may be initiated following tissue injury. METHODS: Pregnant, time-dated mice underwent laparotomy and hysterotomy on embryonic day 15 (E15) and 18 (E18). Full-thickness, excisional cutaneous wounds were made on the dorsum of the fetuses and dorsal skin harvested 15 and 45 min after wounding. Unwounded dorsal skin from additional fetuses collected at the same time points served as controls. The skin was processed to obtain protein, then levels of caspase 3, caspase 7, and poly ADP-ribose polymerase (PARP) were measured by Western blot. Cyclophilin levels were measured to ensure equal loading of protein. Histone-associated DNA complex formation was examined to provide further evidence of cellular apoptosis. RESULTS: There were no differences in total caspase 3 levels between E15 and E18 fetal tissue with or without wounding, nor was any cleavage of caspase 3 noted in any group. However, cleaved caspase 7 was present in the E15 skin with a >2-fold increase following wounding at both 15 and 45 min, yet absent in the E18 groups. Furthermore, levels of cleaved PARP were also increased by >2-fold at both 15 and 45 min in E15 wound groups, whereas a relatively small amount was only seen in the E18 wound groups at 45 min. DNA-histone fragmentation ELISA assay showed a 5-fold increase in the enrichment of histone-associated DNA fragments in the E15 wounded tissue compared with the time-matched controls at 45 min. This was not seen with the E18 tissue. CONCLUSIONS: Previously, we demonstrated that cutaneous wounds in E15 fetal mice heal in a scarless manner, while similar wounds in E18 mice heal with scar formation. Results from our current work demonstrate differences in apoptosis in mid- compared with late-gestational mouse skin as well as shortly after wounding. Our results suggest that in mid-gestational wounds, activation of apoptotic pathways may be mediated through effector caspase 7 signals with inactivation of PARP. This initiation of apoptotic signals following tissue injury may play a role in scarless wound repair.

Differential expression of procollagen genes between mid- and late-gestational fetal fibroblasts.

BACKGROUND: Previously, we have shown that cutaneous wounds in mid-gestational (E15) mice heal in a scarless manner with decreased procollagen 1 and increased procollagen 3 production compared with wounds in late-gestational (E18) mice, which heal with scars. The aim of the current work was to determine whether E15 and E18 fibroblasts respond to stimulation in culture with differential procollagen expression, suggesting they may preserve their phenotype in vitro. Further, we wanted to determine if fetal fibroblast gene expression patterns persisted in tissue culture. We measured expression of procollagen types 1alpha1 and 3 in response to TGF-beta1 stimulation. We theorized that E15 fibroblasts would respond with a pattern of procollagen that would contribute to a more easily remodeled collagen. METHODS: Mid- and late-gestational fetal fibroblasts were obtained from dorsal skin harvested from fetuses of time-dated CD-1 mice. Cells were grown to confluence in culture plates overnight. Cell monolayers were treated with 0.01% bovine serum albumin (BSA) plus 10 ng/well of TGF-beta1. Cells were harvested at 6 and 24 h following treatment. Additional groups were treated with BSA alone (vehicle controls) and collected at 6 and 24 h. Another group without treatment was harvested after reaching confluence (0 time point). In a separate experiment to determine if gene expression patterns persisted, cells were treated with 0.01% BSA plus 10 ng/well of TGF- beta1 for 24 h, then harvested. A second group of cells were treated again at 24 h and harvested at 48 h. Additional cells were treated with BSA alone for 24 and 48 h, and another group without treatment was harvested after reaching confluence (0 time point). Cells were processed to obtain mRNA, cDNA was made, and then samples analyzed by QPCR. Results were analyzed by ANOVA and Holm-Sidak method. RESULTS: Procollagen 1alpha1 gene expression was decreased in E15 cells at 6 and 24 h following TGF-beta1 treatment, P<0.05. In contrast, procollagen 1alpha1 was increased in E18 cells, P<0.05. Procollagen 3 gene expression was decreased in E18 cells at 6 and 24 h following treatment with TGF-beta1, P<0.05, whereas levels in E15 cells were unchanged at 6 h, and only trended lower at 24 h. We evaluated whether this differential expression of procollagen 3 persisted at 24 and 48 h. At 24 and 48 h, E15 control groups had increased procollagen 3 expression compared with E18 groups, P<0.05. E15 and E18 cells in TGF-beta1-treated groups had decreased procollagen 3 at 48h compared with their respective BSA control groups, P<0.05. However, the degree of difference appeared to be greater in the E15 group than the E18 group. CONCLUSIONS: Our results from this in vitro work demonstrate a differential pattern of gene expression for procollagen 1alpha1 and 3 in E15 and E18 fibroblasts in response to TGF-beta1. E15 cells showed decreased expression of procollagen 1alpha1, while E18 cells showed increased procollagen 1alpha1 and decreased procollagen 3 expression. These patterns of expression in E15 cells are suggestive of increased type 3 to 1 collagen ratio seen in scarless fetal wounds. Interestingly, treatment of either E15 or E18 cells with TGF-beta1 significantly decreased procollagen 3 expression by 48 h, yet this was more profound in E15 groups. This suggests that after 24 h, E15 cells may transition towards an E18 phenotype and corresponding signaling.

Homeobox genes Hoxd3 and Hoxd8 are differentially expressed in fetal mouse excisional wounds.

BACKGROUND: Cell signaling pathways underlying wound repair are under extensive investigation; however, there is still a poor understanding of the mechanisms orchestrating these processes. Hox genes, which are a subgroup of homeobox genes, encode for a family of transcription factors that play a critical role in tissue migration and cell differentiation during embryogenesis and may also serve as master regulatory genes of postnatal wound repair. We have developed a fetal excisional wound healing model whereby mid-gestational wounds heal in a regenerative manner while late-gestational wounds display scar formation. We theorize that Hoxd3 and Hoxd8 will be differentially expressed in mid- and late-gestational wounds compared with normal skin. MATERIALS AND METHODS: Pregnant FVB mice underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional wounds were made on the dorsum of each fetus. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control (c) skin samples were also obtained from unwounded adjacent fetuses. Samples were harvested at 3 and 6 h and real-time polymerase chain reaction was performed for Hoxd3 and Hoxd8 and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by analysis of variance with statistical significance of P < 0.05. RESULTS: Hoxd3 levels were increased in all of the mid-gestational groups, with a significant increase at 3 h compared with late-gestational control groups. In the 3-h time group, Hoxd8 is increased in mid-gestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group, where Hoxd8 is increased in mid-gestational wounds compared with all groups. Also, Hoxd8 in the mid-gestational near wound controls is significantly greater than that in the late-gestational near wound control and control groups. CONCLUSIONS: These data suggest that Hoxd3 is constitutively expressed in the skin of mid-gestational mice. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair.