Faculty Bibliography

OBJECTIVE:The authors hypothesized that new agents such as BioGlue would be as efficacious as kaolin in the induction of hydrocephalus in fetal sheep. METHODS:This study was performed in 34 fetal lambs randomly divided into 2 studies. In the first study, fetuses received kaolin, BioGlue (2.0 mL), or Onyx injected into the cisterna magna, or no injection (control group) between E85 and E90. In the second study, fetuses received 2.0-mL or 2.5-mL injections of BioGlue into the cisterna magna between E85 and E90. Fetuses were monitored using ultrasound to assess lateral ventricle size and progression of hydrocephalus. The fetuses were delivered (E120-E125) and euthanized for histological analysis. Selected brain sections were stained for ionized calcium binding adaptor 1 (Iba1) and glial fibrillary acidic protein (GFAP) to assess the presence and activation of microglia and astroglia, respectively. Statistical comparisons were performed with Student's t-test for 2 determinations and ANOVA 1-way and 2-way repeated measures for multiple determinations. RESULTS:At 30 days after injection, the lateral ventricles were larger in all 3 groups that had undergone injection than in controls (mean diameter in controls 3.76 ± 0.05 mm, n = 5). However, dilatation was greater in the fetuses injected with 2 mL of BioGlue (11.34 ± 4.76 mm, n = 11) than in those injected with kaolin (6.4 ± 0.98 mm, n = 7) or Onyx (5.7 ± 0.31 mm, n = 6) (ANOVA, *p ≤ 0.0001). Fetuses injected with 2.0 mL or 2.5 mL of BioGlue showed the same ventricle dilatation but it appeared earlier (at 10 days postinjection) in those injected with 2.5 mL. The critical threshold of ventricle dilatation was 0.1 for all the groups, and only the BioGlue 2.0 mL and BioGlue 2.5 mL groups exceeded this critical value (at 30 days and 18 days after injection, respectively) (ANOVA, *p ≤ 0.0001). Moderate to severe hydrocephalus with corpus callosum disruption was observed in all experimental groups. All experimental groups showed ventriculomegaly with significant microgliosis and astrogliosis in the subventricular zone around the lateral ventricles. Only kaolin resulted in significant microgliosis in the fourth ventricle area (ANOVA, *p ≤ 0.005). CONCLUSIONS:The results of these studies demonstrate that BioGlue is more effective than Onyx or kaolin for inducing hydrocephalus in the fetal lamb and results in a volume-related response by obstructive space-occupancy without local neuroinflammatory reaction. This novel use of BioGlue generates a model with potential for new insights into hydrocephalus pathology and the development of therapeutics in obstructive hydrocephalus. In addition, this model allows for the study of acute and chronic obstructive hydrocephalus by using different BioGlue volumes for intracisternal injection.

PURPOSE:Endoscopic third ventriculostomy (ETV) has become the method of choice in the treatment of hydrocephalus. Age and etiology could determine success rates (SR) of ETV. The purpose of this study is to assess these factors in pediatric population. METHODS:Retrospective study on 51 children with obstructive hydrocephalus that underwent ETV was performed. The patients were divided into three groups per their age at the time of the treatment: < 6, 6-24, and > 24 months of age. All ETV procedures were performed by the same neurosurgeon. RESULTS:Overall SR of ETV was 80% (40/51) for all etiologies and ages. In patients < 6 months of age SR was 56.2% (9/16), while 6-24 months of age was 88.9% (16/18) and > 24 months was 94.1% (16/17) (p = 0.012). The highest SR was obtained on aqueductal stenosis. SR of posthemorrhagic, postinfectious, and spina bifida related hydrocephalus was 60% (3/5), 50% (1/2), and 14.3% (1/7), respectively. While SR rate at the first ETV attempt was 85.3%, it was 76.9% in patients with V-P shunt performed previously (p = 0.000). CONCLUSIONS:Factors indicating a potential failure of ETV were young age and etiology such as spina bifida, other than isolated aqueductal stenosis. ETV is the method of choice even in patients with former shunting. Fast healing, distensible skulls, and lower pressure gradient in younger children, all can play a role in ETV failure. Based on our experience, ETV could be the first method of choice for hydrocephalus even in children younger than 6 months of age.

OBJECTIVE:The purpose of this study was to examine differences between patients with myelomeningocele and those with myelocele with respect to brain imaging findings at fetal MRI. MATERIALS AND METHODS:A single-center retrospective analysis was performed of fetal MRI examinations revealing open spinal dysraphism from 2004 through 2016 with available diagnostic postnatal spinal MR images in conjunction with neurosurgical follow-up findings. Images were reviewed by two board-certified fellowship-trained pediatric neuroradiologists. Relevant clinical data were recorded. RESULTS:The study included 119 fetal MRI examinations of patients with open spinal dysraphism. Myeloceles were found in 29.4% (35/119) of these examinations and myelomeningoceles in the others. All (35/35) myeloceles showed grade 3 (severe) Chiari II malformations. Only 73.8% (62/84) of myelomeningoceles showed grade 3 Chiari II malformation. Clinically significant spinal kyphosis was found in 5.0% (6/119) of fetuses, and all of these fetuses had grade 3 Chiari II malformations. The size of the spinal dysraphic defect had significant positive correlation with lateral (p < 0.0001) and third (p = 0.006) ventricular size. Mean volume of the myelomeningocele sac was significantly different among Chiari II grades and inversely proportional to Chiari II grade (p = 0.0009). CONCLUSION:Larger spinal dysraphic defects correlated with increased ventricular size at fetal MRI. All of the fetuses with myelocele or kyphosis had severe Chiari II malformations. Larger myelomeningocele sac size was associated with lower grade of Chiari II malformation, suggesting that myelomeningocele sac formation may be protective against hindbrain herniation.

BACKGROUND:Fetal tracheal occlusion (TO) is an emerging surgical therapy in congenital diaphragmatic hernia that improves the fetal lung growth. Different animal models of congenital diaphragmatic hernia and TO present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and available genetic tools. We developed a minimally invasive murine transuterine TO model, which will be useful in defining how TO impacts lung molecular biology, cellular processes, and overall lung physiology. MATERIALS AND METHODS:Time-mated C57BL/6 mice underwent laparotomy at embryonic day 16.5 (E16.5) with transuterine TO performed on two fetuses in each uterine horn. At E18.5, dams were sacrificed and fetuses harvested. The lungs of the TO fetuses were compared with the nonmanipulated counterparts by morphometric and histologic analysis. RESULTS:Successful TO was confirmed in 16 of 20 TO fetuses. Twelve of them survived to E18.5 (75%). Fetal weights were comparable, but lung weights were significantly greater in TO (28.41 ± 5.87 versus 23.38 ± 3.09, P = 0.043). Lung to body weight ratio was also greater (0.26 ± 0.003 versus 0.22 ± 0.002, P = 0.006). E18.5 TO lungs demonstrated dilated central and distal airspaces with increased cellularity. DNA/protein and DNA/lung weight ratios were elevated while protein/lung weight ratio was lower in TO compared to control. CONCLUSIONS:Mice fetal transuterine TO is feasible with comparable outcomes to other current animal models. The increase in the lung weight, lung to body weight ratio and the DNA/protein ratio indicate organized lung growth rather than edema or cell hypertrophy.

Spina bifida aperta is a congenital malformation characterized by the failure of neural tube closure resulting in an unprotected fetal spinal cord. The spinal cord then undergoes progressive damage, likely due to chemical and mechanical factors related to exposure to the intrauterine environment. Astrogliosis in exposed spinal cords has been described in animal models of spina bifida during embryonic life but its relationship with neuroinflammatory processes are completely unknown. Using a retinoic acid-induced rat model of spina bifida we demonstrated that, when exposed to amniotic fluid, fetal spinal cords showed progressive astrogliosis with neuronal loss at mid-gestation (E15) compared to unexposed spinal cords. The number of microglial cells with a reactive phenotype and activation marker expression increased during gestation and exhibited progressive disruption in the inhibitory immune ligand-receptor system. Specifically we demonstrate down-regulation of CD200 expression and up-regulation of CD200R. Exposed spinal cords demonstrated neuroinflammation with increased tissue water content and cytokine production by the end of gestation (E20), which correlated with active Caspase3 expression in the exposed layers. Our findings provide new evidence that microglia activation, including the disruption of the endogenous inhibitory system (CD200-CD200R), may participate in the pathogenesis of spina bifida through late gestation.

OBJECTIVE:To compare endotracheal fluid (EF) and amniotic fluid (AF) phospholipidic profile changes following tracheal occlusion (TO) in the congenital diaphragmatic hernia (CDH) fetal lamb model, in order to support the efficacy of TO on lung maturity. METHODS:A diaphragmatic defect was induced at 70 days' gestation, TO was carried out at day 102 and cesarean section at 136 days' gestation. EF and AF samples, collected at delivery, were evaluated using mass spectrometry (the analysis focused on palmitoyloleoyl-phosphatidylcholine [POPC, PC(18:1/16:0)], dipalmitoyl-phosphatidylcholine [DPPC, PC(16:0/16:0)] and sphingomyelins [SMs]). RESULTS:The effects of CDH and TO were different on AF and EF. POPC levels were higher than DPPC levels in AF of healthy lambs. Following induction of the diaphragmatic malformation, an evident decrease in POPC was noted, while a substantial return to normal POPC levels and an increased DPPC peak were prompted by the TO. After CDH induction, a decrease in N-palmitoyl-D-sphingomyelin [SM(d18:1/16:0)] was revealed (P<0.01) and an increased peak in SMs in AF was prompted by the TO (P=0.05). While the most represented phosphatidylcholine (PC) species in EF of healthy lambs was DPPC, CDH induced a decrease in the DPPC peak and treatment with TO induced its partial recovery. SMs were detectable only in healthy EF samples. CONCLUSION/CONCLUSIONS:The phospholipid recovery profile following TO suggests the potential role of this therapy in restoring processes involved in surfactant-mediated lung maturation, even though other interactions involved in AF turnover should be considered. Moreover, these metabolites could be used as biomarkers of fetal pulmonary development.

OBJECTIVE:To describe development of a two-port fetoscopic technique for spina bifida repair in the exteriorized, carbon dioxide-filled uterus and report early results of two cohorts of patients: the first 15 treated with an iterative technique and the latter 13 with a standardized technique. METHODS:This was a retrospective cohort study (2014-2016). All patients met Management of Myelomeningocele Study selection criteria. The intraoperative approach was iterative in the first 15 patients and was then standardized. Obstetric, maternal, fetal, and early neonatal outcomes were compared. Standard parametric and nonparametric tests were used as appropriate. RESULTS:Data for 28 patients (22 endoscopic only, four hybrid, two abandoned) are reported, but only those with a complete fetoscopic repair were analyzed (iterative technique [n=10] compared with standardized technique [n=12]). Maternal demographics and gestational age (median [range]) at fetal surgery (25.4 [22.9-25.9] compared with 24.8 [24-25.6] weeks) were similar, but delivery occurred at 35.9 (26-39) weeks of gestation with the iterative technique compared with 39 (35.9-40) weeks of gestation with the standardized technique (P<.01). Duration of surgery (267 [107-434] compared with 246 [206-333] minutes), complication rates, preterm prelabor rupture of membranes rates (4/12 [33%] compared with 1/10 [10%]), and vaginal delivery rates (5/12 [42%] compared with 6/10 [60%]) were not statistically different in the iterative and standardized techniques, respectively. In 6 of 12 (50%) compared with 1 of 10 (10%), respectively (P=.07), there was leakage of cerebrospinal fluid from the repair site at birth. Management of Myelomeningocele Study criteria for hydrocephalus-death at discharge were met in 9 of 12 (75%) and 3 of 10 (30%), respectively, and 7 of 12 (58%) compared with 2 of 10 (20%) have been treated for hydrocephalus to date. These latter differences were not statistically significant. CONCLUSION:Fetoscopic open neural tube defect repair does not appear to increase maternal-fetal complications as compared with repair by hysterotomy, allows for vaginal delivery, and may reduce long-term maternal risks. CLINICAL TRIAL REGISTRATION:ClinicalTrials.gov, https://clinicaltrials.gov, NCT02230072.

BACKGROUND:Skeletal muscle injuries are the most common sports-related injury and a major concern in sports medicine. The effect of platelet-rich plasma (PRP) injections on muscle healing is still poorly understood, and current data are inconclusive. PURPOSE/OBJECTIVE:To evaluate the effects of an ultrasound-guided intramuscular PRP injection, administered 24 hours after injury, and/or posttraumatic daily exercise training for 2 weeks on skeletal muscle healing in a recently established rat model of skeletal muscle injury that highly mimics the muscle trauma seen in human athletes. STUDY DESIGN/METHODS:Controlled laboratory study. METHODS:A total of 40 rats were assigned to 5 groups. Injured rats (medial gastrocnemius injury) received a single PRP injection (PRP group), daily exercise training (Exer group), or a combination of a single PRP injection and daily exercise training (PRP-Exer group). Untreated and intramuscular saline-injected animals were used as controls. Muscle force was determined 2 weeks after muscle injury, and muscles were harvested and evaluated by means of histological assessment and immunofluorescence microscopy. RESULTS:Both PRP (exhibiting 4.8-fold higher platelet concentration than whole blood) and exercise training improved muscle strength (maximum tetanus force, TetF) in approximately 18%, 20%, and 30% of rats in the PRP, PRP-Exer, and Exer groups, respectively. Specific markers of muscle regeneration (developmental myosin heavy chain, dMHC) and scar formation (collagen I) demonstrated the beneficial effect of the tested therapies in accelerating the muscle healing process in rats. PRP and exercise treatments stimulated the growth of newly formed regenerating muscle fibers (1.5-, 2-, and 2.5-fold increase in myofiber cross-sectional area in PRP, PRP-Exer, and Exer groups, respectively) and reduced scar formation in injured skeletal muscle (20%, 34%, and 41% of reduction in PRP, PRP-Exer, and Exer groups, respectively). Exercise-treated muscles (PRP-Exer and Exer groups) had significantly reduced percentage of dMHC-positive regenerating fibers (35% and 47% decrease in dMHC expression, respectively), indicating that exercise therapies accelerated the muscle healing process witnessed by the more rapid replacement of the embryonic-developmental myosin isoform by mature muscle myosin isoforms. CONCLUSION/CONCLUSIONS:Intramuscular PRP injection and, especially, treadmill exercise improve histological outcome and force recovery of the injured skeletal muscle in a rat injury model that imitates sports-related muscle injuries in athletes. However, there was not a synergistic effect when both treatments were combined, suggesting that PRP does not add any beneficial effect to exercise-based therapy in the treatment of injured skeletal muscle. CLINICAL RELEVANCE/CONCLUSIONS:This study demonstrates the efficacy of an early active rehabilitation protocol or single intramuscular PRP injection on muscle recovery. The data also reveal that the outcome of the early active rehabilitation is adversely affected by the PRP injection when the two therapies are combined, and this could explain why PRP therapies have failed in randomized clinical trials where the athletes have adhered to postinjection rehabilitation protocols based on the principle of early, active mobilization.

Despite benefits of prenatal in utero repair of myelomeningocele, a severe type of spina bifida aperta, many of these patients will still suffer mild to severe impairment. One potential source of stem cells for new regenerative medicine-based therapeutic approaches for spinal cord injury repair is neural progenitor cells (NPCs) in cerebrospinal fluid (CSF). To this aim, we extracted CSF from the cyst surrounding the exposed neural placode during the surgical repair of myelomeningocele in 6 fetuses (20 to 26weeks of gestation). In primary cultured CSF-derived cells, neurogenic properties were confirmed by in vitro differentiation into various neural lineage cell types, and NPC markers expression (TBR2, CD15, SOX2) were detected by immunofluorescence and RT-PCR analysis. Differentiation into three neural lineages was corroborated by arbitrary differentiation (depletion of growths factors) or explicit differentiation as neuronal, astrocyte, or oligodendrocyte cell types using specific induction mediums. Differentiated cells showed the specific expression of neural differentiation markers (βIII-tubulin, GFAP, CNPase, oligo-O1). In myelomeningocele patients, CSF-derived cells could become a potential source of NPCs with neurogenic capacity. Our findings support the development of innovative stem-cell-based therapeutics by autologous transplantation of CSF-derived NPCs in damaged spinal cords, such as myelomeningocele, thus promoting neural tissue regeneration in fetuses.

OBJECTIVE:To evaluate the independent association of fetal pulmonary response and prematurity to postnatal outcomes after fetal tracheal occlusion for congenital diaphragmatic hernia. METHODS:Fetal pulmonary response, prematurity (<37 weeks at delivery) and extreme prematurity (<32 weeks at delivery) were evaluated and compared between survivors and non-survivors at 6 months of life. Multivariable analysis was conducted with generalized linear mixed models for variables significantly associated with survival in univariate analysis. RESULTS:Eighty-four infants were included, of whom 40 survived (47.6%) and 44 died (52.4%). Univariate analysis demonstrated that survival was associated with greater lung response (p=0.006), and the absence of extreme preterm delivery (p=0.044). In multivariable analysis, greater pulmonary response after FETO was an independent predictor of survival (aOR 1.87, 95% CI 1.08-3.33, p=0.023), whereas the presence of extreme prematurity was not statistically associated with mortality after controlling for fetal pulmonary response (aOR 0.52, 95% CI 0.12-2.30, p=0.367). CONCLUSION/CONCLUSIONS:Fetal pulmonary response after FETO is the most important factor associated with survival, independently from the gestational age at delivery.