David Lanning

Regulation of fibroblast functions by lysophospholipid mediators : Potential roles in wound healing

The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P), are recent additions to the list of potent mediators of tissue repair and wound healing. In this review, we highlight the diverse actions of LPA and S1P on many types of cells involved in the wound healing process, with special emphasis on their regulation of fibroblasts. The effects of LPA and S1P are principally mediated via specific cell surface receptors. Important signaling pathways downstream of these receptors and the importance of TGFβ and S1P cross‐talk for wound healing are also discussed. Moreover, specific agonists and antagonists of the lysophospholipid receptors may be useful for the treatment of wounds and abnormal wound healing.

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.

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.

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 childrens 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.

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.

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.

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.

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.

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.

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.