Visible light-curable methacrylated glycol chitosan hydrogel patches for prenatal closure of fetal myelomeningocele.

In this study, we prepared visible light-curable methacrylated glycol chitosan (MGC) hydrogel patches for the prenatal treatment of fetal myelomeningocele (MMC) and investigated their feasibility using a retinoic acid-induced fetal MMC rat model. 4, 5, and 6 w/v% of MGC were selected as candidate precursor solutions, and photo-cured for 20 s, because the resulting hydrogels were found to possess concentration dependent tunable mechanical properties and structural morphologies. Moreover, these materials exhibited no foreign body reactions with good adhesive properties in animal studies. The inflammation scoring assessment in vivo exhibited the absence of foreign body reactions in MGC hydrogel treated lesion. The complete epithelial coverage of MMC was made with using 6 w/v% MGC hydrogel followed by well-organized granulation along with noticeable decrease of abortion rate and wound size that highlight the therapeutic potential for the prenatal treatment of fetal MMC.

Intrauterine neuromuscular and stromal dysplasia of the bladder in retinoic acid-induced myelomeningocele fetal rats.

OBJECTIVES: To observe the changes in the bladder of fetal rats with myelomeningocele (MMC) induced by all-trans retinoic acid (atRA) during the embryonic development stages. METHODS: The fetal rat model of MMC was induced by intragastric administration of atRA to pregnant rats on embryonic day 10 (E10). Fetal rats were harvested at E16, E18, E20, and E21 for observation and further testing. Those with MMC were classified as the MMC group, while those without MMC as the RA group. The areas of MMC skin defect, the crown-rump length (CRL), and body weight in different groups were compared. The histopathological changes in the bladder were compared. The expression levels of alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMMHC), connexin 43 (Cx43), desmin, ß3 tubulin, and vesicular acetylcholine transporter (VAChT) in the bladder were investigated by immunohistochemical staining and Western blotting. Pregnant rats given intragastric administration with olive oil (OIL group) at E10 were set as the blank control group. RESULTS: A total of 415 fetal rats of different gestational ages were harvested with an MMC incidence of 56.05 % (139/248). The incidence rate increased with embryonic days (p < 0.001). Compared with the other two control groups, the CRL and bodyweight of MMC fetal rats were significantly delayed at E21 (p < 0.001). The expression levels of αSMA, SMMHC, Cx43, desmin, ß3 tubulin and VAChT in the bladder of MMC fetal rats were significantly decreased at E21 (p < 0.05). CONCLUSIONS: In atRA-induced MMC fetal rats, there is neural, muscular, and stromal dysplasia in the bladder at an early gestational age. Further investigations on neurogenic bladder secondary to MMC are applicable using this animal model.

Intra-amniotic Injection of Poly(lactic-co-glycolic Acid) Microparticles Loaded with Growth Factor: Effect on Tissue Coverage and Cellular Apoptosis in the Rat Model of Myelomeningocele.

BACKGROUND: Myelomeningocele (MMC) is a devastating congenital neurologic disorder that can lead to lifelong morbidity and has limited treatment options. This study investigates the use of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with fibroblast growth factor (FGF) as a platform for in utero treatment of MMC. STUDY DESIGN: Intra-amniotic injections of PLGA MPs were performed on gestational day 17 (E17) in all-trans retinoic acid-induced MMC rat dams. MPs loaded with fluorescent dye (DiO) were evaluated 3 hours after injection to determine incidence of binding to the MMC defect. Fetuses were then treated with PBS or PLGA particles loaded with DiO, bovine serum albumin, or FGF and evaluated at term (E21). Fetuses with MMC defects were evaluated for gross and histologic evidence of soft tissue coverage. The effect of PLGA-FGF treatment on spinal cord cell death was evaluated using an in situ cell death kit. RESULTS: PLGA-DiO MPs had a binding incidence of 86% and 94% 3 hours after injection at E17 for doses of 0.1 mg and 1.2 mg, respectively. Incidence of soft tissue coverage at term was 19% (4 of 21), 22% (2 of 9), and 83% (5 of 6) for PLGA-DiO, PLGA-BSA, and PLGA-FGF, respectively. At E21, the percentage of spinal cord cells positive for in situ cell death was significantly higher in MMC controls compared with wild-type controls or MMC pups treated with PLGA-FGF. CONCLUSION: PLGA MPs are an innovative minimally invasive platform for induction of soft tissue coverage in the rat model of MMC and may reduce cellular apoptosis.

Amylase concentration and activity in the amniotic fluid of fetal rats with retinoic acid induced myelomeningocele.

BACKGROUND: In utero neurologic injury in myelomeningocele (MMC) occurs via a two-hit process: failed neural tube closure followed by neurodegeneration in utero. Meconium in the amniotic fluid contains pancreatic digestive enzymes and is neurotoxic in rat models of MMC. OBJECTIVES: The objectives of this study were to demonstrate the neurotoxicity of α-amylase and to compare the enzyme concentration and activity in the amniotic fluid of rats with retinoic acid induced MMC to a healthy control population. STUDY DESIGN: Timed pregnant Sprague Dawley rats were gavage fed all-trans retinoic acid (60 mg/kg) in olive oil on gestational day E10 to induce a MMC defect. Control rats received olive oil. Amniotic fluid was collected on embryonic days E15, E17, E19, and E21. The amniotic fluid amylase concentration and relative activity were measured at each gestational age, and levels were compared between the MMC and control groups using Wilcoxon Rank Sum and Kruskal-Wallis tests. In a subset of dams sacrificed on E10.5, neuroepithelial cells were isolated from control embryos and exposed to α-amylase in increasing concentrations. Percentage of cell survival was assessed with CellProfiler software. RESULTS: Amniotic fluid amylase activity for embryonic days E15, E17, E19, and E21 was determined for MMC and control pups. Amylase activity increased significantly from E15 to E21 in both control (p = 3.0 × 10-5) and MMC (p = 1.5 × 10-5) groups. Relative amylase activity was significantly increased in MMC pups compared to controls on E19 (247,792.8 versus 106,263.6; p = .0019) and E21 (772,645.8 versus 481,975.3; p = .021); no difference was detected on E15 (36,646.8 versus 40,179.3; p = .645) or E17 (121,617.5 versus 71,750; p = 1.000). In vitro, amylase demonstrated dose-dependent toxicity to fetal rat neuroepithelial cells. CONCLUSION: Amylase concentration and activity level were higher in the amniotic fluid of rats with retinoic acid induced MMC compared to controls with advancing gestational age. As amylase is toxic to neural epithelial cells, the higher activity of this digestive enzyme in fetuses with MMC may be a contributor to neural tube damage in utero. Future research should focus on amylase and other digestive enzymes in human MMC, as they may serve as potential targets of in utero therapy.

Preliminary Evaluation of a Novel Fetal Guinea Pig Myelomeningocele Model.

INTRODUCTION: Translational models of myelomeningocele (MMC) are needed to test novel in utero interventions. An ideal animal model for MMC has locomotor function at birth and is low cost enough to allow for high throughput. The rat MMC model is limited by immature locomotor function at birth. The ovine MMC model is a costly surgical model. Guinea pigs are uniquely suited for an MMC model being a small animal model with locomotor function at birth. We aimed to develop a retinoic acid (RA) model of MMC in the guinea pig and to evaluate if pregnant guinea pigs could tolerate uterine manipulation. METHODS: Time-mated Dunkin Hartley guinea pig dams were dosed with 60 mg/kg of RA between gestation age (GA) 12 and 15 days in the development of an RA model. Fetuses were grossly evaluated for MMC lesions at Cesarean section after GA 31 days. Evaluation of the ability of pregnant guinea pig dams to tolerate uterine surgical intervention was performed by hysterotomy of a separated group of time-mated guinea pigs at GA 45, 50, and 55. RESULTS: Forty-two pregnant guinea pigs were dosed with RA, with a total of 189 fetuses. The fetal demise rate was 38% (n = 71). A total of 118 fetuses were viable, 83% (n = 98) were normal fetuses, 8% (n = 10) had a neural tube defect, and 8% (n = 10) had a hematoma or other anomalies. No fetuses developed an MMC defect. None of the fetuses that underwent hysterotomy survived to term. CONCLUSION: RA dosed at 60 mg/kg in guinea pigs between GA 12 and 15 did not result in MMC. Dunkin Hartley guinea pigs did not tolerate a hysterotomy near term in our surgical model. Further work is needed to determine if MMC can be induced in guinea pigs with alternate RA dosing.

Diffusion weighted imaging as a biomarker of retinoic acid induced myelomeningocele.

Neural tube defects are a common congenital anomaly involving incomplete closure of the spinal cord. Myelomeningocele (MMC) is a severe form in which there is complete exposure of neural tissue with a lack of skin, soft tissue, or bony covering to protect the spinal cord. The all-trans retinoic acid (ATRA) induced rat model of (MMC) is a reproducible, cost-effective means of studying this disease; however, there are limited modalities to objectively quantify disease severity, or potential benefits from experimental therapies. We sought to determine the feasibility of detecting differences between MMC and wild type (WT) rat fetuses using diffusion magnetic resonance imaging techniques (MRI). Rat dams were gavage-fed ATRA to produce MMC defects in fetuses, which were surgically delivered prior to term. Average diffusion coefficient (ADC) and fractional anisotropy (FA) maps were obtained for each fetus. Brain volumes and two anatomically defined brain length measurements (D1 and D2) were significantly decreased in MMC compared to WT. Mean ADC signal was significantly increased in MMC compared to WT, but no difference was found for FA signal. In summary, ADC and brain measurements were significantly different between WT and MMC rat fetuses. ADC could be a useful complementary imaging biomarker to current histopathologic analysis of MMC models, and potentially expedite therapeutic research for this disease.

In Utero Amniotic Fluid Stem Cell Therapy Protects Against Myelomeningocele via Spinal Cord Coverage and Hepatocyte Growth Factor Secretion.

Despite the poor prognosis associated with myelomeningocele (MMC), the options for prenatal treatments are still limited. Recently, fetal cellular therapy has become a new option for treating birth defects, although the therapeutic effects and mechanisms associated with such treatments remain unclear. The use of human amniotic fluid stem cells (hAFSCs) is ideal with respect to immunoreactivity and cell propagation. The prenatal diagnosis of MMC during early stages of pregnancy could allow for the ex vivo proliferation and modulation of autologous hAFSCs for use in utero stem cell therapy. Therefore, we investigated the therapeutic effects and mechanisms of hAFSCs-based treatment for fetal MMC. hAFSCs were isolated as CD117-positive cells from the amniotic fluid of 15- to 17-week pregnant women who underwent amniocentesis for prenatal diagnosis and consented to this study. Rat dams were exposed to retinoic acid to induce fetal MMC and were subsequently injected with hAFSCs in each amniotic cavity. We measured the exposed area of the spinal cord and hepatocyte growth factor (HGF) levels at the lesion. The exposed spinal area of the hAFSC-treated group was significantly smaller than that of the control group. Immunohistochemical analysis demonstrated a reduction in neuronal damage such as neurodegeneration and astrogliosis in the hAFSC-treated group. Additionally, in lesions of the hAFSC-treated group, HGF expression was upregulated and HGF-positive hAFSCs were identified, suggesting that these cells migrated to the lesion and secreted HGF to suppress neuronal damage and induce neurogenesis. Therefore, in utero hAFSC therapy could become a novel strategy for fetal MMC. Stem Cells Translational Medicine 2019;8:1170-1179.

Altered Amniotic Fluid Levels of Hyaluronic Acid in Fetal Rats with Myelomeningocele: Understanding Spinal Cord Injury.

Myelomeningocele (MMC) is a devastating congenital neural tube defect that results in the exposure of spinal cord to the intrauterine environment, leading to secondary spinal cord injury and severe impairment. Although the mechanisms underlying the secondary pathogenesis are clinically relevant, the exact cause of in utero-acquired spinal cord damage remains unclear. The objective of this study was to determine whether the hyaluronic acid (HA) concentration in amniotic fluid (AF) in the retinoic acid-induced model of MMC is different from that in normal controls and whether these differences could have an impact on the viscosity of AF. Our data shows that the concentration of HA in AF samples from fetuses with MMC (MMC-AF) and normal control samples (Norm-AF) were not significantly different at embryonic day 18 (E18) and E20. Thereafter, the HA concentration significantly increased in Norm-AF but not in MMC-AF. Compared with Norm-AF, the concentration of HA in MMC-AF and the viscosity of MMC-AF were significantly lower at E21. Agarose gel electrophoresis confirmed a significant reduction in the HA level of MMC-AF compared with Norm-AF at E21. No HA-degrading activity was detected in MMC-AF. In summary, we identified a deficiency in the AF level of HA and the viscosity of AF in fetal rats with MMC. These data are discussed in relation to a potential role the reduction in the AF viscosity due to the low level of HA may play in the exacerbating effects of mechanical trauma on spinal cord damage at the MMC lesion site.

Amniotic fluid levels of phospholipase A2 in fetal rats with retinoic acid induced myelomeningocele: the potential "second hit" in neurologic damage.

OBJECTIVES: There is growing evidence of ongoing, in utero neurological damage in fetuses with myelomeningocele (MMC). Phospholipase A2 (PLA2) has known neurotoxic properties and is predominantly present in its secretory isoform (sPLA2) in meconium, the passage of which is increased in MMC fetuses. The objective of this study was to determine if amniotic fluid (AF) levels of PLA2 are elevated in a rat model of MMC. METHODS: Timed pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid (RA) in olive oil at embryonic day 10 (E10). Amniocentesis was performed at multiple gestational time points on MMC fetuses, RA-exposed fetuses without MMC and control fetuses. AF PLA2 levels were analyzed by a fluorescent enzyme activity assay. PLA2 isoforms were determined by measuring activity in the presence of specific inhibitors. RESULTS: There was no difference in AF PLA2 activity between groups on E15. PLA2 activity was significantly increased in MMC fetuses on E17, E19 and E21 (p < 0.001). Secretory PLA2 primarily accounted for the overall greater activity. CONCLUSIONS: PLA2 levels are elevated in the AF of fetal rats with MMC and may contribute to ongoing neural injury. This pathway may be a useful drug target to limit ongoing damage and better preserve neurologic function.

Fetal Myelomeningocele After Maternal Methotrexate Administration: A Case Report.

Background: Folate supplementation in women of reproductive age has a well-established role in the prevention of neural tube defects. Methotrexate is a commonly used drug which functions by inhibiting normal folate metabolism in active cells. An association between fetal methotrexate exposure and myelomeningocele might be expected, considering this relationship. However, to our knowledge, no cases of myelomeningocele secondary to in utero methotrexate exposure have been reported. Case: We present the case of a gravid patient who, having received methotrexate for management of an ectopic pregnancy, was lost to follow-up and returned several weeks later carrying an intrauterine pregnancy. The fetus was found prenatally to be suffering from multiple congenital anomalies. At birth the infant demonstrated many of the abnormalities commonly associated with fetal methotrexate syndrome, including craniosynostosis and talipes equinovarus. Most interestingly, the newborn was also diagnosed with a lumbar myelomeningocele and concomitant type II Chiari malformation, as is often associated with such a neural tube defect. Conclusion: Methotrexate exposure may impact the fetal risk of myelomeningocele. Patients should be counseled thoroughly on the importance of follow-up care.

Arsenic is associated with reduced effect of folic acid in myelomeningocele prevention: a case control study in Bangladesh.

BACKGROUND: Arsenic induces neural tube defects in several animal models, but its potential to cause neural tube defects in humans is unknown. Our objective was to investigate the associations between maternal arsenic exposure, periconceptional folic acid supplementation, and risk of posterior neural tube defect (myelomeningocele) among a highly exposed population in rural Bangladesh. METHODS: We performed a case-control study that recruited physician-confirmed cases from community health clinics served by Dhaka Community Hospital in Bangladesh, as well as local health facilities that treat children with myelomeningocele. Controls were selected from pregnancy registries in the same areas. Maternal arsenic exposure was estimated from drinking water samples taken from wells used during the first trimester of pregnancy. Periconceptional folic acid use was ascertained by self-report, and maternal folate status was further assessed by plasma folate levels measured at the time of the study visit. RESULTS: Fifty-seven cases of myelomeningocele were identified along with 55 controls. A significant interaction was observed between drinking water inorganic arsenic and periconceptional folic acid use. As drinking water inorganic arsenic concentrations increased from 1 to 25 µg/L, the estimated protective effect of folic acid use declined (OR 0.22 to 1.03), and was not protective at higher concentrations of arsenic. No main effect of arsenic exposure on myelomeningocele risk was identified. CONCLUSIONS: Our study found a significant interaction between drinking water inorganic arsenic concentration from wells used during the first trimester of pregnancy and reported intake of periconceptional folic acid supplements. Results suggest that environmental arsenic exposure reduces the effectiveness of folic acid supplementation in preventing myelomeningocele.

Micro-computed tomography assessment of vertebral column defects in retinoic acid-induced rat model of myelomeningocele.

BACKGROUND: Myelomeningocele (MMC) is a common congenital malformation and the most severe form of spina bifida characterized by the protrusion of spinal cord and meninges through the spinal defect. Our objective was to improve the assessment of congenital vertebral defects in animal models of MMC using three-dimensional high resolution micro-computed tomography (micro-CT) imaging and quantitative digital analyses methods. METHODS: Lumbosacral MMC was induced in fetal rats by exposure of pregnant mothers at embryonic day 10 (E10) to all-trans retinoic acid, and rats were examined at term (embryonic day 22). The axial skeleton was examined in an MMC model for the first time using ex vivo micro-CT at 10 µm voxel resolution to allow high resolution two-dimensional and three-dimensional characterization of anomalies in lumbosacral vertebrae, and quantitative assessment of distances between dorsal vertebral arches in lumbosacral regions in MMC rats, compared with normal controls. RESULTS: We observed, in detail, skeletal defects in lumbosacral vertebra of MMC rats, including in the morphology of individual dorsal vertebral arches. Use of high resolution micro-CT has also enabled us to identify the delayed (nonfused) or absent ossification in vertebral bodies, increased fusion of adjacent lateral vertebral elements, and quantify the extent of dorsal arch widening. Distances between dorsal vertebral arches showed statistically significant increases from L5 through S4 in MMC rats, compared with normal controls. CONCLUSION: High-resolution micro-CT combined with digital quantification methods is a powerful technique ideally suited for precise assessment of complex congenital skeletal abnormalities such as examined in this rodent model of MMC.

Reduction in neural injury with earlier delivery in a mouse model of congenital myelomeningocele: laboratory investigation.

OBJECT: The authors undertook this study to assess the effect of preterm delivery with respect to neural protection in a congenital myelomeningocele (MMC) mouse model. METHODS: After confirmation of pregnancy in 15 female mice, a congenital MMC model was produced by administration of retinoic acid on the 7th day of gestation. The pregnant mice underwent cesarean sections on Days 15 (n = 5, Group E15), 17 (n = 5, Group E17), and 19 (n = 5, Group E19). Histological analyses were conducted on the lumbar defect and on the craniocervical junction in all fetuses with MMC. RESULTS: Fetuses in Group E19 showed the most significant injury to neural tissue of the spinal cord at the MMC area followed by those in Group E17, with Group E15 being the least affected. All groups exhibited a degree of Chiari malformation; Group E19 was the most affected, followed by Group E17, and Group E15 was the least affected. CONCLUSIONS: Development of both Chiari malformation and exposed spinal cord injury are progressive during gestation. Preterm delivery in this mouse model of congenital MMC may minimize the degree of injury to the spinal cord neural tissue and the degree of Chiari malformation.

Neurotoxic effect of povidone-iodine on the rat spine using a laminectomy-durotomy model.

OBJECTIVE: The efficacy and safety of povidone-iodine in wound dressing and irrigation of some operative cavities were established by many in vitro and in vivo experimental reports and clinical series. However, its use in defective tissue in neural structures has not been confirmed yet. The aim of the present study was to histopathologically investigate its effect on neural tissues when applied on the upper side of defective dura. METHODS: Wistar rats were randomly divided into two experimental groups: control and povidone-iodine groups. In the control group, durotomy was performed following laminectomy, and the spinal cord was covered with a dry sponge. In the study group, the same procedure was performed, but open duras were covered with a sponge that had been wetted with 0.1 % povidone-iodine solution. Three weeks after surgery, all experimental animals were sacrificed, and histopathological evaluations were conducted. RESULTS: Myelin changes were absent or minimal in all cases of the control group but were present as markedly increased myelin degeneration in nearly all cases in the study group. Axonal degeneration and hypoxic neuronal damage were absent in the control group, whereas they were marked in half of the study group. No statistically significant differences were established in Schwann cell proliferation, venous congestion, and lymphocytic proliferation between the two groups. CONCLUSIONS: Based on the present study, 0.1 % povidone-iodine solution cannot be recommended for wound dressing for neural structures such as myelomeningocele cases because of possible damage to underlying neural tissues.

Increased hepatic Igf2 gene expression involves C/EBPß in TCDD-induced teratogenesis in rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant for reproductive toxicity that was suggested to be linked to growth factors. Insulin-like growth factor 2 (Igf2) has great effects on the control of fetal growth. We hypothesize it might participate in the TCDD-induced toxic events. The expression of Igf2 in TCDD-induced fetal rat and rat hepatoma BRL-3A cells was monitored by real-time quantitative RT-PCR and Western blotting. Electrophoresis mobility shift assay and chromatin immunoprecipitation were performed to identify the CCAAT/enhancer binding protein ß (C/EBPß) responsive element in the Igf2 P3-promoter. The transcriptional activity of the Igf2 P3-promoter was detected by luciferase assay. Pregnant rats exposed to TCDD showed a modest incidence of fetal death, fetal growth restriction and fetal malformation. The levels of Igf2 mRNA and IGF2 protein were elevated in TCDD-exposed fetal liver. Temporal expression of Igf2 was also induced by TCDD in BRL-3A cells. A C/EBPß responsive element was identified at position -743 to -732 of the Igf2 P3-promoter, and its binding was enhanced by TCDD exposure through upregulation of the C/EBPß protein. The transcriptional activity of the Igf2 P3-promoter was also augmented by TCDD. Our results showed that TCDD may induce Igf2 gene expression through the transactivation of C/EBPß, which may be linked to the developmental effects of TCDD in rats.

Amniotic fluid levels of glial fibrillary acidic protein in fetal rats with retinoic acid induced myelomeningocele: a potential marker for spinal cord injury.

OBJECTIVE: The objective of this study was to determine whether amniotic fluid levels of glial acidic fibrillary protein (GFAP) would reflect myelomeningocele-related neurodegeneration in the rat model of retinoic acid-induced myelomeningocele, which is a model that is very similar to human myelomeningocele and develops the entire spectrum of disease severity including features of the Chiari II malformation. STUDY DESIGN: Time-dated (embryonic day 10) pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid that had been dissolved in olive oil or olive oil alone. Myelomeningocele, retinoic acid-exposed no myelomeningocele, and control fetuses were harvested at specific time points throughout gestation. A standard set of pinching tests was performed to interrogate the sensorimotor reflex arc of hindpaws and tails. Amniotic fluid-GFAP levels were analyzed by standard enzyme-linked immunosorbent assay techniques. RESULTS: Amniotic fluid-GFAP levels were similar between groups at embryonic days 14, 16, and 18, respectively. Compared with control fetuses, amniotic fluid GFAP levels were significantly increased in myelomeningocele fetuses at embryonic days 20 and 22 (P < .001). Defect size (P < .001), presence of clubfoot deformity (P = .0004), and absence of sensorimotor function (P < .01) at embryonic day 22 correlated with amniotic fluid-GFAP levels. CONCLUSION: Amniotic fluid-GFAP levels appear to correlate with spinal cord injury as gestation proceeds in fetal rats with myelomeningocele.

Sodium valproate and the fetus: a case study and review of the literature.

Sodium valproate is a teratogen responsible for a wide range of abnormalities, including neural tube defects. It has traditionally been prescribed for epilepsy, but is increasingly used for such psychiatric conditions as bipolar disease. Women of childbearing age taking valproate should be warned of its teratogenicity and advised to plan pregnancies, take a higher dose of folate, discuss reducing the dose of valproate or changing the medication with their physician, and have antenatal screening. After birth, the infant should be examined for a wide range of reported abnormalities. Neurodevelopmental assessment should continue throughout childhood. We present a case that illustrates the need for better education of mothers taking valproate and the medical staff prescribing it.

Morphologic analysis of the neuromuscular development of the anorectal unit in fetal rats with retinoic acid induced myelomeningocele.

To investigate whether myelomeningocele (MMC) is associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution and staining intensity of non-neuronal (alpha-smooth-muscle-actin), neural crest cell (NCC, [Hoxb5]), and neuronal markers (PGP-9.5, synaptophysin, neurotubulin-beta-III) within the distal colon, rectum, and anal sphincters were analyzed by immunohistochemistry and Western blot in rat fetuses with retinoic acid (RA) induced MMC. At term (E22), no gross-morphological differences of the anorectal unit of OIL (n=21) MMC (n=31), and RA-exposed-non MMC (RA, n=19) fetuses were found. Smooth muscle cells were evenly distributed within the muscle layers of the rectum and the internal anal sphincter in OIL, MMC, and RA fetuses. Density and staining intensity of NCC and mature enteric neurons within the myenteric plexus of the distal colon and rectum and innervation pattern within anal sphincters in MMC fetuses were analogous to RA and OIL controls. Normal smooth muscle and myenteric plexus development of the rectum and normal innervation of the anal sphincters and pelvic floor suggests that MMC is not associated with a global neuromuscular maldevelopment of lower GI structures in this short-gestational model.

Structural and functional characterization of bladder smooth muscle in fetal rats with retinoic acid-induced myelomeningocele.

Myelomeningocele (MMC) is the most common cause of neurogenic bladder dysfunction (NBD). We recently developed a novel retinoic acid (RA)-induced MMC model in fetal rats. The objective of this study was to use this model to assess functional and structural characteristics of the detrusor muscle in MMC-associated NBD. Time-dated pregnant Sprague-Dawley rats were gavage fed 60 mg/kg RA dissolved in olive oil or olive oil alone [embryonic day 10 (E10)]. Bladder specimens from olive oil-exposed fetuses (OIL; n = 71), MMC (n = 79), and RA-exposed-no MMC (RA, n = 62) were randomly assigned for functional and histopathological evaluation and protein analysis. Contractility responses to field and agonist-mediated stimulation (KCl and bethanecol) were analyzed. The expression patterns of alpha-smooth muscle actin, myosin, desmin, vimentin, and collagen III and I were analyzed by immunohistochemistry and Western blotting. Spatial and temporal distribution of nerve fibers within the detrusor muscle was monitored by neurotubulin-beta-III throughout gestation. Neither OIL, MMC, nor RA detrusor responded to field stimulation. MMC bladder strips showed a significant decrease in contractility after KCl and bethanechol stimulation compared with OIL and RA bladders. Bladder detrusor morphology and expression patterns of smooth muscle markers were similar between groups. Detrusor muscles in OIL and RA fetuses were densely innervated, possessing abundant intramural ganglia and nerve trunks that branch to supply smooth muscle bundles. In MMC bladders, neurotubulin-beta-III-positive nerve fibers were markedly decreased with advancing gestational age and were almost completely absent at term (E22). We conclude that the biomechanical properties of fetal rat MMC bladders are analogous to that seen in humans with MMC-associated NBD. Decreased nerve density indicates loss of peripheral neural innervation throughout gestation. The early observation of decreased innervation and decreased contractility in the absence of morphologic abnormalities in muscle structure or extracellular matrix supports a pathophysiological hypothesis that denervation is the primary insult preceding the observed alterations in bladder muscle structure and function.