https://doi.org/10.1007/s00381-021-05299-1 INVITED PAPER
State of the art in translating experimental myelomeningocele research to the bedside
Lourenço Sbragia1 · Karina Miura da Costa1 · Antonio Landolffi Abdul Nour1 · Rodrigo Ruano2 · Marcelo Volpon Santos3 · Hélio Rubens Machado3
Received: 15 July 2021 / Accepted: 18 July 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
Myelomeningocele (MMC), the commonest type of spina bifida (SB), occurs due to abnormal development of the neural tube and manifest as failure of the complete fusion of posterior arches of the spinal column, leading to dysplastic growth of the spinal cord and meninges. It is associated with several degrees of motor and sensory deficits below the level of the lesion, as well as skeletal deformities, bladder and bowel incontinence, and sexual dysfunction. These children might develop varying degrees of neuropsychomotor delay, partly due to the severity of the injuries that affect the nervous system before birth, partly due to the related cerebral malformations (notably hydrocephalus—which may also lead to an increase in intracranial pressure—and Chiari II deformity). Traditionally, MMC was repaired surgically just after birth; however, intrauterine correction of MMC has been shown to have several potential benefits, including better sensorimotor outcomes (since exposure to amniotic fluid and its consequent deleterious effects is shortened) and reduced rates of hydrocephalus, among others. Fetal surgery for myelomeningocele, nevertheless, would not have been made possible without the develop‑
ment of experimental models of this pathological condition. Hence, the aim of the current article is to provide an overview of the animal models of MMC that were used over the years and describe how this knowledge has been translated into the fetal treatment of MMC in humans.
Keywords Experimental myelomeningocele · Fetal surgery · Myelomeningocele · Spina bifida
Myelomeningocele (MMC), meningocele, and spina bifida (SB) constitute a spectrum of spinal dysraphisms that are characterized as a defect in the development of the neural tube and manifest as failure in the complete fusion of the posterior arches of the spinal column, leading to dysplastic growth of the spinal cord and meninges [1]. Their incidence in the USA is approximately 1 for every 1000 live births [2]. The diagnosis of fetal MMC is frequently performed by
ultrasonography or magnetic resonance imaging in routine prenatal scans, after 16 weeks of gestation [3].
MMC is associated with several degrees of motor and sensory deficits below the level of the lesion, as well as skeletal deformities, bladder and bowel incontinence, and sexual dysfunction. Hydrocephalus is usually considered the most severe associated alteration and occurs, among other pathophysiological conditions, alongside type II Chiari mal‑
formation (CM), which consists of a complex anomaly of the posterior fossa, characterized by downward displacement of the medulla oblongata and cerebellum through the foramen magnum into the cervical spinal canal. These children might develop varying degrees of neuropsychomotor delay, partly due to the severity of the injuries that affect the nervous sys‑
tem before birth, partly due to the related cerebral malforma‑
tions, and partly due to an increase in intracranial pressure [4, 5]. Consequently, the average longevity of patients born with MMC is reduced to less than 40 years, and they also experience a considerably worse quality of life, in addition to enormous personal, familiar, and social consequences [6].
* Hélio Rubens Machado hrmachad@fmrp.usp.br
1 Division of Pediatric Surgery – Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
2 Division of Maternal‑Fetal Medicine, Department
of Obstetrics and Gynecology, University of Texas, Houston, TX, USA
3 Division of Pediatric Neurosurgery – Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
/ Published online: 31 July 2021
The estimated cost of health care for myelomeningocele patients, in 1985, was more than 200 million dollars per year, only in the USA [7]. Furthermore, even with the stand‑
ard surgical treatment, approximately 14% of neonates with MMC do not survive beyond the age of 5, bringing this mortality rate to around 35% in those with dysfunction of the brain stem secondary to CM malformation [8]. So far, intrauterine correction of MMC has the potential to limit the progression of hydrocephalus, ergo reducing the need for ventriculo‑peritoneal shunts (VPS) and its consequent complications, which are not infrequent [9].
The first experimental MMC model with fetal surgical repair was devised by Michejda [10] in primates, consisting of a group of animals which was immediately operated after production of the spinal defect with allogeneic bone paste, and a control group not submitted to repair. The latter group developed neurologi‑
cal deficits and cystic lesions of the spinal cord similar to those found in humans [11]. These initial studies were followed by the introduction of the toxicogenic rat model of MMC induced by retinoic acid [12], and, as a result, the experimental models of MMC have brought remarkable contributions which were ultimately transcribed to the clinical setting. In this review, we report the different experimental models of MMC that have been used over the years and describe how this knowledge might be translated into the fetal treatment of MMC in babies.
Myelomeningocele in chicken embryos
Surgical models of spina bifida in chicksA summary of the main contributions provided by experimen‑
tal models of spina bifida in chicken can be found in Table 1.
This model was first developed by Rokos et al. (1973), who split the spinal cord of incubated 3‑day‑old chicks, via open‑
ing of the eggshell, removal of the paper‑thin membrane below the air chamber, and topical application of 1–3 drops of neutral red. Section of the spinal cord was carried out under direct observation using a stereomicroscope. The incision over
the posterior roof of the central canal was up to eight somites long, with a sharp needle. Histological evaluation at 1, 2, and 3–4 h post‑operatively confirmed adequate opening of the spi‑
nal cord [13]. Mann and Persaud [14] described that window‑
ing alone is enough to cause neural tube defects. Open brain defects and myeloschisis were observed and accompanied by extensive cystic and hemorrhagic changes in the adjacent mesoderm, along with reduction in somite volume [14].
Pilowsky et al. [15] induced neural tube defects by remov‑
ing albumen, disrupting the closed neural tube mechanically, or injecting tetanus toxin. All three resulted in increased acetyl‑
cholinesterase concentration in the amniotic fluid when com‑
pared to controls, with no difference in concentration between the three teratogenic methods. They suggested that determining acetylcholinesterase concentration in the amniotic fluid could be translated into clinical practice to detect neural tube defects [15].
Lopez de Torre et al. [16] described the induction of neu‑
ral tube defects (NTDs) by aspiration of albumen. After 24 h of incubation, eggs were punctured, and 5 ml of albumen were aspirated, and incubation resumed. Nearly half (45%) of the embryos survived, with 12% of them presenting with some sort of malformation, half of those being NTDs, such as open myelomeningocele, meningocele, and encephalocele [16]. Clark and Scothorne [17] described the effects of a neu‑
ral tube incision in different stages of the Hamburger and Hamilton scale (chicks hatch after 21 days of incubation).
Embryos operated at earlier stages (12/13) had an almost 90% rate of healing of the defect. This rate fell to 43%, 19%, and 0% when the incision was made on embryos at stages 14, 15, and 16, respectively. When the defect persisted, its length also showed a correlation to the stage, with later stages showing larger defects than earlier ones [17] (Figs. 1 and 2).
Olguner et al. [18] compared the effect of amniotic fluid replacement and neural tissue damage in chick embryos with myelomeningocele. The defect was created on 13‑day‑old incubated embryos, using a midline incision, posterior lami‑
nectomy, and opening of the dura. In one of the groups, the amniotic fluid was exchanged with an iso‑osmolar solution, controlling the amount of replacement fluid according to
Table 1 Translational contributions to the study of MMC in the chick model. SBA, spina bifida aperta
Author Year Contribution
Rokos and Knowles [13] 1976 Opening of the neural tube with needle and induction of defect Mann and Persaud [14] 1979 Windowing as etiology of neural tube defects
Pilowsky et al. [15] 1982 Increased acetylcholinesterase in amniotic fluid
Lopez de Torre et al. [16] 1990 Induction of neural tube defects by aspiration of albumen Clark and Scothorne [17] 1990 Effect of neural tube incision on different developmental stages Olguner et al. [18] 2000 Effect of amniotic fluid replacement
Mominoki et al. [11] 2006 Diminished interneurons in injured spinal cord Wang et al. [19] 2011 Migration and development of motor neurons Tsujimura et al. [20] 2011 Sensory tract abnormalities
Khan et al. [21] 2017 Neuronal and neurotransmitter changes in SBA motor neurons
the expected volume on the control group. On the 18th day of incubation, embryos were harvested and examined. Both groups showed an open dorsal defect with exposure of the spinal cord. Microscopically, the exchange group showed mild edema, calcification, fibrosis, capillary cell prolifera‑
tion, and presence of mononuclear cells, when compared to the MMC‑only group. Furthermore, the number of neuron‑
specific enolase (NSE) stained neurons was significantly higher in the exchange group [18].
Mominoki et al. [11] investigated lower limb function in a surgical model of spina bifida aperta (SBA) in chicks.
As expected, chicks with SBA had dysfunctional legs, with impaired locomotion capability. Upon microscopical analysis of the lesional areas, they noted a specific decrease in small neurons (50–400 μm2), which likely represent interneurons, while the number of larger neurons was not affected. They hypothesized that this could be due to the absence of signaling proteins, factors normally secreted by the closed neural tube and necessary for the generation of distinct dorsal interneurons [11, 19]. studied the develop‑
ment of motor neurons in chicks with SBA. Using an anti‑
body against islet‑1, a motor neuron marker, they described the presence of such neurons in the lesional area of embryos in different embryonic stages and compared them with chronologically equivalent controls. Although the number of islet‑1 positive motor neurons was equivalent in the final
embryonic days (day 6 onward), a delay in migration from the ependymal layer to the ventral horn and in maturation, as reflected by increased extracellular spaces between neurons, was noticed on the SBA group [19].
Tsujimura et al. [20] studied sensory tracts in the surgi‑
cal model of spina bifida in chicks described by Mominoki.
Operated chicks had a smaller dorsal funiculus, with smaller dimensions and less nerve fibers than controls. Furthermore, the group described, in a section at the level of the dorsal root ganglia of L5, degeneration of fibers in the ipsilateral and contralateral dorsal, ventral, and lateral funiculi of the spinal cord, in contrast to the degeneration of fibers only in the ipsilateral dorsal funiculus of control chicks, which represents an anomaly of the upward sensory nerve fibers in SBA [20].
Khan et al. [21], also working with a surgical model of spina bifida, found that loss of voluntary leg movements coincides with changes in neuronal networks at the lesion site [21]. Firstly, the number of motor neurons seems to be preserved during the embryonic period and early neona‑
tal days (embryonic day 14 to postnatal day (PD) 4) but decreased by PD10. There was also a correspondent reduc‑
tion in the gray matter of the motor neuron region. Electron microscopy was then used to evaluate the synaptic altera‑
tions that occurred in the motor neuron region. It revealed that, in severely symptomatic SBA chicks, significant
Fig. 1 Surgical procedure in the chick embryo
Fig. 2 Postnatal defect in the chick embryo (left); creation of the defect, through an open eggshell (right)
detachment of synaptic boutons had occurred by PD10.
Quantitative analysis revealed that the total number of syn‑
aptic boutons per neuron in SBA chicks did not differ from that of controls on PD0, PD2, or PD4. The SBA chicks had, however, a greater number of excitatory boutons and a reduced number of inhibitory boutons.
To better understand the inhibitory and excitatory changes in motor neurons and whether they began before or after hatching, the same authors utilized immunostaining for choline acetyltransferase (ChAT) and glutamic acid decar‑
boxylase 67 (GAD67), from ED18 to PD10. SBA chicks showed increased immunoreactivity already at ED18, while in controls, the expression of the markers increased only after hatching. Furthermore, in accordance with the elec‑
tron microscopy assessments, SBA animals had increased ChAT and reduced GAD67 expression on PD2 and PD4, and by PD10 both had decreased, with an almost absent GAD67 labeling. The authors also performed immunostain‑
ing for apoptosis marker caspase 3, from ED18 to PD10.
The expression of such protein was weak at ED18, moderate to high at PD2 and PD4, and weak at PD10; this last result probably reflecting near‑total degeneration of GABAergic interneurons. Similar results were observed when caspase 3 expression was studied on motor neurons, but with stronger expression remaining on PD10. To clarify the origin of the described GABAergic, cholinergic and apoptotic changes, SBA chicks were compared to sham counterparts, with neural tube incisions of less than three somites, and post‑
natal sham groups, in which the defect was made on PD0 by means of a posterior laminectomy from L2 to L4. None of the sham groups showed spinal cord deformation or the aforementioned GABA, ChAT, or caspase 3 expression, sug‑
gesting that the changes observed in SBA chicks were likely the result of spinal cord exposure to amniotic fluid [21].
Myelomeningocele in mice
The curly tail modelThe ct mutant mouse provides a reliable model for the analy‑
sis of neural tube defects (NTD). The ct gene has variable expression and is incompletely penetrant, with homozygotes developing exencephaly in around 3% of cases, spina bifida aperta in 10%, and a curled tail in 50% [8]. This strain, first described by Grünenberg et al. [22], consists of a genetic model passed on by a recessive gene (ct) that arose as a spontaneous mutation [22, 23]. Adinolfi et al. [24] found increased alpha‑fetoprotein levels in the amniotic fluid of ct fetuses with exencephaly and open spina bifida, corroborat‑
ing the validation of this model for human defects [24].
Seller [25] reported that maternal administra‑
tion of intraperitoneal hydroxyurea, mitomycin C, or
5‑fluorouracil on gestational day (GD) 9 of ct pregnant mice led to a significant reduction in the proportion of fetal NTD [25]. The same group published in 1986 that, according to the development stage in which mitomycin C was administered, it might either have a teratogenic (GD 7) or preventive effect (GD 9) [26]. Copp and Brook [27], studying this model, concluded that SBA occurs initially as a defect of primary neurulation, and that fundamental dif‑
ferences in the mechanisms of primary neurulation in the cranial and lower spinal regions of mammalian embryos can account for the gender‑related differences observed in the incidence of NTD affecting these levels of the body axis (female embryos are more likely to develop exenceph‑
aly while lower spinal defects are more common in males) [27]. Brook et al. [28] proposed that the defect occurs in the ct model because an increased ventral curvature of the neuropore region imposes a mechanical stress, which coun‑
terbalances neurulation and thus delays or prevents closure of the posterior neuropore [28].
Selçuki et al. [29] showed that early neuronal differ‑
entiation in the ct model appears intact within the MMC.
They point out that, of all the available mouse models, this is the one that most closely resembles non‑syndromic human NTD comprising anencephaly and lumbosacral MMC in the absence of other birth defects [29]. Petzold et al. [30] used this model to analyze whether brain‑
specific proteins would enable monitoring of the develop‑
ment of MMC‑related tissue damage during pregnancy.
Their findings suggest that spinal cord damage starts to expedite around gestational day 16.5, and axonal degen‑
eration is more severe in large MMC defects [30]. These findings were supported by the study of Stiefel et al. [31], who found intact exposed spinal cord in early gestation and progressive destruction of the unprotected neural tis‑
sue subsequently [32].
Reis et al. [33] showed that neural degeneration in this model involves progressive loss of neuronal cells and an increase in astrocytes, supporting the potential benefits of surgical treatment of MMC infants in utero [33]. These results were in accordance with the ones of Stiefel et al., who also found increasing neurodegeneration within the SB lesion, paralleled by a progressive loss of neurological function [34]. Reis et al. [3], in addition, found increased vascular density and apoptosis at late stages of gestation in MMC, supporting that in utero changes of the placode, particularly during these last stages of gestation, contrib‑
ute to the neuropathological manifestations seen in full‑
term newborns with MMC [3]. Also, using this model, Bardill et al. [35] injected reverse thermal gel over the spinal defect, which formed a stable layer covering the MMC and was not associated with inflammation, making this technique a promising minimally invasive therapeutic approach [35].
The curtailed model
Searle [36] described a dominant mutation induced by radia‑
tion in the T‑locus (gene symbol Tc) that resulted in a tailless phenotype and occult SB [36]. When Tc is heterozygous with certain recessive mutations such as tw5 (Tc/Tw5), a more severe phenotype ensues, thus providing an animal model to study the mechanisms that induce SB with MMC [37, 38].
Splotch
Dickie et al. [39] described the splotch (Spd) mouse, a semi‑dominant mutation that originated spontaneously in the C‑57 BL/6J mouse strain, and whose homozygote phenotype (Spd/Spd) includes lumbosacral SB [39, 40].
McLone et al. [41] studied this model to look for evidence of abnormalities of neural tissue exposed to amniotic fluid but found no evidence of destruction of the exposed neural tube [41]. Liu et al. [2] assessed whether anorectal malfor‑
mations and anterior sacral MMC share the same embryo‑
genic pathway by administering etretinate to Spd mice on GD 9. In this study, they established the mouse model of the Currarino triad of congenital caudal anomalies: ano‑
rectal malformations, sacral abnormalities, and presacral mass [2].
Drug‑induced defects
Ehlers et al. [42] investigated the relationship between val‑
proic acid (VPA) administration and the development of SB in Han:NMRI strain mice and showed that the injection of VPA on GD 9 results in a low incidence of open SB and a high incidence of occult SB [42]. Kochhar [43] described
the occurrence of occult SB in pregnant A/Jax mice when 2 mg of retinoic acid (RA) was administered by gavage on GD 9 [43]. Yasuda et al. [44] found SB in ICR strain mice exposed to 60 mg/kg of oral RA on GD 8 [44]. Tibbles and Wiley [45] reported myeloschisis in CD‑1 mice gavage fed with 80 mg/kg of RA on GD 9 [45].
Seller et al. [46] described a paradoxical effect of RA on the phenotypic expression of the ct mutation: when low doses (5 mg/kg) were given intraperitoneally on GD 9, rather than GD 8, the incidence of NTD was decreased to a level even lower than that found spontaneously in the ct strain [46]. This was supported by subsequent studies [26, 47].
Quemelo et al. [48] administered 70 mg/kg of intraperito‑
neal RA to Swiss mice on GD 7 and 8, finding higher rates of exencephaly, MMC, and occult SB when the drug was given on GD 8 [48]. Al‑Shanafey et al. [49] gavage fed RA to C57BL/6 J pregnant mice on GD 7 to study the effect of preterm delivery on neural protection and found that it can possibly minimize the degree of injury to the spinal cord neural tissue and the development of Chiari deformity type II [49].
Surgical model
Inagaki et al. [50] operated on pregnant mice at GD 12 (time of mice pregnancy = 19 days), using a sharp needle to split the spinal cord dorsally, sectioning the roof of the neural tube for approximately 2–3 somites at the thoracic and/or lumbar region. Using this methodology, they supported the hypothesis that open neural tube defects allow for cerebro‑
spinal fluid (CSF) leakage and cause brain abnormalities due to reduced intraventricular CSF pressure [50]. A timeline of mouse models and their respective contributions is displayed in Table 2.
Table 2 Translational contributions to the study of MMC of the mice model
*Currarino triad: anorectal malformations, sacral abnormalities, and presacral mass
Authors Year Contribution
Grünenberg [22] 1954 Description of the curly tail model
Dickie [39] 1964 Description of the splotch model
Searle [36] 1966 Description of the curtailed model
Kochhar [43] 1967 Occurrence of MMC with administration of retinoic acid Adinolfi et al. [24] 1976 Increased alpha‑fetoprotein levels in the amniotic fluid
of fetuses with SB
Seller et al. [46] 1983 Effect of hydroxyurea, mitomycin C, and 5‑fluorouracil on the development of NTDs
Ehlers et al. [42] 1992 Valproic acid–induced defect
Inagaki et al. [50] 1997 Description of the surgical model of MMC
Liu et al. [2] 2003 Established the model of the Currarino triad in mice*
Petzold et al. [30] 2005 Progressive destruction of neural tissue during gestation Bardill et al. [35] 2019 Use of reverse thermal gel to cover the MMC
Myelomeningocele in rats
The trypan blue modelThe first MMC rat model was described by Gillman et al.
[51] and consisted of a subcutaneous injection of 1 cc of 1.0% trypan blue into female rats before conception and/
or during pregnancy. The model was not ideal because pups had a low incidence of the spinal malformation (6.1%;
43/697) [51]. Peters and de Geus [52] supported the hypoth‑
esis of indirect teratogenic effect of the trypan blue upon the embryo, interfering with the function of the extra‑embryonic membranes and the primitive gut and resulting in caudal MMC [52].
The surgical model
Heffez et al. [53] created MMC surgically in Sprague‑Dawley rats at GD 18 (time of rat pregnancy = 21 days) (Fig. 3) to test the hypothesis that paralysis may be due in part to a spi‑
nal cord injury caused by exposure of the neural tube to the amniotic fluid and proposed a “two‑hit” hypothesis to explain motor deficits seen in children with MMC: (1) congenital myelodysplasia complicated by (2) intrauterine spinal cord injury [53]. The same group (1993) reported the results of intrauterine treatment in this model, which suggested that both mechanical trauma and toxic injury may contribute to spinal cord dysfunction [54].
Drewek et al. [55] assessed the toxic effects of human amniotic fluid of various gestational ages upon organotypic cultures of rat spinal cord and found that it became toxic at approximately 34 weeks of gestation [55]. Correia‑Pinto et al. [56] found increased inflammation and delayed repair processes in meconium‑exposed dysraphic spinal cords of rat fetuses [56]. Weber Guimarães Barreto et al. [57] found that there is a high incidence of Chiari malformation in this rat model [57]. The same group (2009) evaluated the effect
of steroid therapy on neurological disabilities and reported significant clinical improvement after treatment [58].
The all‑trans retinoic acid model
Danzer et al. [12] proposed another toxicologic model for MMC in rats using gavage‑fed RA at GD 10 of Sprague‑
Dawley pregnant rats. This model resulted in a 60.7% rate (307/505) of isolated MMC in exposed fetuses [12] (Fig. 4).
The same group (2007) assessed functional and struc‑
tural characteristics of the detrusor muscle in rats with MMC and associated neurogenic bladder and concluded that the biomechanical properties of fetal MMC bladders in the rat are analogous to those seen in humans [59]. They suggested that MMC was not associated with an impaired global neuromuscular development of lower gastroin‑
testinal structures in this model (2008) [60]. Also, these authors showed that glial fibrillary acidic protein (GFAP) levels in the amniotic fluid of this model correlated with spinal cord injury as the gestation proceeded (2011) [61].
Watanabe et al. [62] evaluated the therapeutic feasibil‑
ity of a tissue engineering approach for prenatal coverage of RA‑induced MMC in the rat model with encouraging results [62, 63].
Fig. 3 Surgical creation of MMC in rat fetus (left) and postnatal appearance of the defect (right)
Fig. 4 Toxicological model of MMC with retinoic acid in rats
Pennington et al. [64] suggested that quantitative amni‑
otic cell profiling may become a useful diagnostic tool in the prenatal evaluation of neural tube defects. They stud‑
ied amniotic fluid samples in this model and found that the proportions of neural and mesenchymal stem cells in the amniotic fluid correlate with the type and size of neural tube defects [64].
Shen et al. [65] analyzed the development and inner‑
vation of the smooth muscle of the bladder in correla‑
tion with lesions of the spinal cord at different gestational ages (16, 18, and 20 gestational days) of fetal MMC in rats and found that it seems morphologically normal in developmental terms, whereas its innervation is markedly impaired [65].
Dionigi et al. [6, 66] found that amniotic mesenchy‑
mal stem cells can induce partial or complete coverage of experimental spina bifida after concentrated intra‑amniotic injection and mitigates the occurrence of Chiari II mal‑
formation [6, 66]. They compared placental‑derived and amniotic fluid‑derived mesenchymal stem cells in transam‑
niotic stem cell therapy and found that both can induce comparable rates of coverage of experimental spina bifida [6]. Shieh et al. [67] found that amniotic mesenchymal stem cells engraft to specific sites (placenta, umbilical cord, spleen, bone marrow, hipbones, brain, and the defect itself) due to a hematogenous route as well as distant cen‑
tral nervous system homing [67].
Considering the evidence of ongoing in utero neuro‑
logical damage in fetuses with MMC and the neurotoxic properties of phospholipase A2, [68] described elevated levels of this enzyme in the amniotic fluid and suggested that it may contribute to the second hit of neurologic damage [68]. Tekin et al. [69] studied the bladders of
Wistar albino rats with RA‑induced MMC and found a decreased density of interstitial cells of Cajal [69]. Zieba et al. [70] identified a neuroepithelial cell population in the fluid of MMC fetuses that formed adherent clusters in culture, which were absent from normal control fetuses.
Their data also suggests that the phase of the disease is a crucial factor in the appearance of these cells, and this may provide an important platform for studying the progression of MMC and for the development of strate‑
gies of repair and diagnosis of MMC prenatally [70].
They also identified a deficiency of hyaluronic acid in the amniotic fluid and discussed the potential role of reduced amniotic fluid viscosity, which may aggravate the effects of mechanical trauma and spinal cord damage at the MMC site [71].
Regarding experimental fetal therapy, Kajiwara et al. [72]
performed antenatal treatment using manufactured three‑
dimensional skin substitutes obtained from induced pluri‑
potent stem cells of a patient. The artificial skin successfully covered the MMC defect when operated on gestational day 20 and harvested at gestational day 22 [72]. Tang et al. [73]
analyzed the effect of fetal MMC repair, using a chitosan‑
gelatin membrane patch, on bladder function. Abnormal neuromuscular development was observed, which enabled a certain degree of improvement after in utero MMC repair [73]. Farrelly et al. [74] injected alginate microparticles loaded with basic fibroblast growth factor in the amniotic fluid at gestational day 17.5, which resulted in significant soft tissue coverage of the MMC defect (Table 3) [74]. Abe et al. [75] used human amniotic fluid stem cells in the amni‑
otic cavity and reported a reduction of the exposed spinal area and thus neuronal damage (as seen per neurodegenera‑
tion and astrogliosis processes) in the treated group [75].
Table 3 Translational contributions of the study of MMC in the rat model
Authors Year Contribution
Gillman et al. [51] 1948 Description of the trypan blue model
Heffez et al. [53] 1990 Creation of the surgical model and proposition of the “two‑hit” hypothesis
Heffez et al. [54] 1993 Intrauterine treatment of the surgical model
Drewek et al. [55] 1997 Amniotic fluid becomes toxic at 34 weeks of gestation Correia‑Pinto et al. [56] 2002 Effect of human meconium on the exposed spinal cord
Danzer et al. [12] 2005 Description of the all‑trans retinoic acid model
Melo‑Filho et al. [58] 2009 Effect of corticosteroid treatment on neurological disabilities
Pennington et al. [64] 2013 Amniotic fluid profile correlates with type and size of neural tube defect
Dionigi et al. [66] 2015 MMC coverage induced by stem cells
Zieba et al. [70] 2017 Examined the cellular content of amniotic fluid
Kajiwara et al. [72] 2017 Used artificial skin in fetal coverage of MMC
Tang et al. [73] 2017 Analyzed chitosan‑gelatin membrane patch for the fetal treatment of MMC Farrelly et al. [74] 2019 Studied the amniotic injection of alginate with fibroblast growth factors
Abe et al. [75] 2019 Used injection of human amniotic fluid stem cells
Myelomeningocele in rabbits
Surgical modelBrunelli and Brunelli [76] first described the surgical manip‑
ulation of rabbit fetuses to create myelomeningocele (MMC) (Table 4). They described open intrauterine surgery, via laparotomy of the pregnant rabbit, identification of the fetus inside the uterus, hysterectomy, and creation of a defect, in which the skin, the posterior vertebral arch, and the menin‑
ges are incised. Upon delivery, no newborn rabbits had med‑
ullary extrusion, and the lesions on the skin, paravertebral muscles, and posterior arch were repaired and showed no evidence of scarring [76]. Calvano et al. [77] described the utilization of laser for the creation of a spinal defect. Dur‑
ing intrauterine surgery, rabbit fetuses were delivered up to the low thoracic level and holmium‑YAG laser was used to perform a L5 to S3 laminectomy vertebrae with preservation of the dura. Subsequent histological analysis of operated fetuses revealed an open defect with intact spinal cord [77].
The first successful surgical creation of MMC in a rab‑
bit model was performed by Housley et al. [78] (Fig. 5).
Pregnant New Zealand white rabbits were operated at GD 22 (time of rabbit pregnancy = 31 days), the fetal dorsum was exposed, and a three‑ or four‑level lumbar laminectomy was made along with dural removal. All injured fetuses were smaller and weighed less than non‑operated counterparts;
histologic examination confirmed a spina bifida‑like lesion, with visible herniation of the cord [78]. Grande et al. [79]
employed evoked potentials in a surgical model of MMC similar to the one described by Housley and demonstrated absent response to stimuli in the lower limbs of animals with spina bifida, whereas the upper limbs and control animals responded normally [79].
Pedreira et al. [80] modified the techniques described by Calvano and Housley. Using intrauterine surgery as well, rabbit fetuses had their dorsum and tail exposed, leading to greater stability during the procedure. In addition, gentle pressure over the insertion of fetal hindlimbs, while incising
the lamina, was used to help to visualize of the spinal cord.
With this technique, once the lamina is incised, the medulla immediately herniates, reducing the risk of cord damage by the surgeon [80]. Repair was attempted using two different techniques. The first consisted of applying a cellulose graft directly to the area where the skin had been removed. The second one consisted of further dissection of the skin defect and placement of the graft underneath the skin, also com‑
pletely covering the defect. None of the repair methods was effective, as demonstrated by the presence of a myelomenin‑
gocele‑like defect in all surviving fetuses.
Juliá et al. [81] demonstrated that covering the fetal spi‑
nal cord decreases neurologic sequelae. Using the model described by Grande et al., they devised three experimen‑
tal groups: control (C), myelomeningocele (M), and mye‑
lomeningocele repaired with an autologous skin flap (T) and evaluated weight and neurological abnormalities (atrophy or deformity of lower limbs, paralysis, response to pain, and ability to climb a 30° slope), as well as the third ven‑
tricular area and somatosensory‑evoked potentials of the limbs. No difference was found between groups M and T on
Table 4 Translational contributions to the study of MMC in the rabbit model. TRASCET, transamniotic stem cell therapy
Authors Year Contribution
Brunelli and Brunelli [76] 1984 First published attempt of surgical creation of spinal defect Calvano et al. [77] 1998 Creation of spinal defect with holmium laser
Housley et al. [78] 2000 First successful surgical creation of MMC
Grande et al. [79] 2002 Absent evoked potential responses in hindlimbs of MMC fetuses Pedreira et al. [80] 2003 Repair attempt with cellulose graft
Juliá et al. [81] 2006 Reduced neurologic sequelae with covering of the defect
Fontecha et al. [82] 2007 Impact of preterm delivery and steroid treatment, defect healing and hindbrain herniation Fontecha et al. [83] 2010 Impact of preterm delivery and steroid treatment on severity of Chiari malformation Shieh et al. [84] 2019 Use of TRASCET in the rabbit model of spina bifida to induce defect coverage with neoskin
Fig. 5 Surgically created MMC in rabbits. Note the exposure of the spinal cord on the left picture
physical examination [81]. Evoked potential amplitudes of hindlimbs were flat on M animals and significantly reduced on T animals, when compared to controls, which were con‑
sidered normal. The latency period analysis revealed similar responses, with controls having the smaller latency period, M groups animals with no evoked potential, and hence, no latency period and T group animals had a significantly greater latency period when compared to controls. The third ventricular area was significantly larger in myelomenin‑
gocele fetuses than in control fetuses, with repaired animals showing values between both, although with no significant difference.
Fontecha et al. [82] hypothesized that preterm delivery and corticosteroid (betamethasone) treatment could have a positive effect on the neural tissue injury caused by amni‑
otic fluid [82]. Four groups were created: MMC, MMC and steroid treatment (MMC‑C), MMC and birth advance‑
ment (MMC‑P), and MMC and steroid treatment and birth advancement (MMC‑CP). Pregnant rabbits were given 0.1 ml/kg of betamethasone in a single dose, after surgery for the creation of MMC. They evaluated limb deformities, grade of kyphosis, vitality, sensibility (pressure required for foot withdrawal), motor evoked potentials (cortical and peripheral), and histological changes in both the MMC and the cervical‑skull zone. After delivery, rabbits treated with steroids or premature birth had fewer deformities, although no difference on kyphosis level was noticed. No difference on vitality and sensibility was observed between groups with MMC.Untreated MMC fetuses had absent neurophysiological signals at all times, which reappeared in newborn rabbits treated only with steroids. On the other hand, no rabbit born prematurely showed motor responses as per evoked poten‑
tials, whether treated with steroids or not. Upon histological study of the MMC zone, newborn rabbits with MMC and no treatment had important spinal cord lesions, with a narrow band of fibrin appearing on the exterior part of the defect and a wide band of neuronal tissue exhibiting progressive cellular destruction. Newborn rabbits treated with steroids prenatally had the most consistent fibrin band as well as some associated cellularity. Underneath, the band of neu‑
ronal tissue was significantly less destroyed. Pups born pre‑
term showed a small band of fibrin and cellular destruction.
Newborn rabbits with both treatments showed mild injury to the neural tissue. Histological examination of the cervical zone revealed milder herniation of the inferior pole of the cerebellum on all treated groups when compared to non‑
treated MMC groups, which was significantly different [82].
Fontecha et al. [83] studied once again the effect of pre‑
term delivery and steroid (betamethasone) treatment; this time regarding alterations associated with the Chiari II mal‑
formation. After creation of a myelomeningocele defect and consecutive histological analysis, the degree of hindbrain
herniation was determined by the percentage of descent of the cerebellar vermis using the distance between the end of the occipital bone and the beginning of the first vertebral arch. The degree of hindbrain herniation was 80% in the not treated group, 36.8% in birth advancement plus steroid treatment, 41.8% in birth advancement, and 44.4% in ster‑
oid treatment. Treated groups showed less severe hindbrain herniation than non‑treated animals, although no significant differences between treated animals were noted [83].
Shieh et al. [84] used heterologous rabbit amniotic mes‑
enchymal stem cells (afMSCs) injected into the amniotic cavity to study the induction of partial or complete skin cov‑
erage of spina bifida. Using intrauterine surgery, a four‑level lumbar laminectomy was performed on fetuses, with subse‑
quent exposure of the distal spinal cord, and no violation of the dura mater. Intra‑amniotic injection of either saline or a suspension of afMSCs was undertaken. Upon histological examination, 50% of the fetuses in the stem cell treatment group had some degree of coverage of the defect, defined as the presence of a rudimentary neoskin with either paucity or complete lack of adnexa. None of them, however, had a complete closure of the defect [84].
Myelomeningocele in sheep
The modelMeuli et al. [85] pioneered the study of MMC in sheep and analyzed whether chronic exposure of the normal spinal cord to amniotic fluid produces a lesion mimicking human MMC.
The defect was created at GD 60 and 75. The first group presented with less severe histological changes and mild paraparesis, while the latter showed MMC‑type pathology much alike humans and paraplegia [85] (Fig. 6).
Bouchard et al. [86] reported that the addition of a myel‑
otomy to this model led to hindbrain herniation similar to that observed in human Chiari II malformation, supporting the hypothesis that leakage of cerebrospinal fluid through the exposed central canal alters normal hydrodynamics and results in herniation, and fetal MMC repair reverses such herniation and restores the gross anatomy of the vermis [86].
Conversely, in the experiments of Guilbaud et al. [87], per‑
forming a myelotomy did not lead to hindbrain herniation in all animals [87].
The evaluation of how closely the surgically induced sheep model resembles the central nervous system derange‑
ments seen in human disease was again the focus of the study from Von Koch et al. [88]. They reported that, although the animal model reproduces the cerebrospinal fluid leak, it does not mimic the developmental defect [88]. Also seeking for model validation, Pedreira et al. [89] described a flattened medulla with disappearance of the medullary canal and
disruption of normal architecture with neuronal apoptosis and/or fusion of the pia mater and dura mater, in addition to herniation of the cerebellum into the cervical canal and syringomyelia. Their conclusion was that the sheep model does mimic the defect found in human fetuses, including the Chiari malformation [89].
Encinas Hernández et al. [90] examined brain malfor‑
mations present in the model, and showed hydrocepha‑
lus, extensive areas of polymicrogyria, denudation of the ependymal lining under polymicrogyric areas, and thinning of the corpus callosum. They did not observe any hindbrain herniation [90]. Amouee et al. [91] evaluated the effects of amniotic fluid on histopathologic changes of the exposed spinal cord of sheep fetuses and described edema, focal cal‑
cification, fibrosis, and capillary cell proliferation [91]. The description of urinary tract malformations in this model was also provided by Encinas et al. [92], who reported on blad‑
ders with urothelial thinning and proliferation of submucosal fibrous tissue [92]. With regard to the variability of innate fetal healing of the defect at the time of repair, Brown et al.
[93] proposed a standardized grading system of the defect based on the percentage of exposed spinal cord and degree of scarring to ensure scientific rigor within the model [93].
They also proposed a standardized rating scale for neuro‑
logic outcomes within seven categories [94]. Galganski et al.
[95] suggested postnatal assessment of spinal angulation to compare functional outcomes of different studies [95]. Steele et al. (2019) evaluated the use of a heritable occurrence of spina bifida in sheep as a natural source for spina bifida fetuses and suggested that it could supplement the draw‑
backs of current models [5].
In utero closure
Copeland et al. [96] described MMC repair in utero using fetal lambs at GD 90 (time of sheep pregnancy = 140 days). The defect and its repair were performed at the same
procedure, using skin grafts obtained from the mother. They found excellent graft coverage and tissue overgrowth over the lesion [96]. Meuli‑Simmen et al. [98] performed closure in utero of the defect with a “reversed” latissimus dorsi flap at GD 100 and demonstrated healed skin wounds, preserved sensory function, absence of incontinence, and near‑normal hindlimb function [97, 98].
Regarding sensory function, Yingling et al. [99] devel‑
oped a technique to record somatosensory evoked potentials in sheep neonates. Animals with MMC showed no soma‑
tosensory evoked potentials after posterior tibial nerve stimulation, but normal potentials after ulnar stimulation.
Surgical repair in utero resulted in preservation of neuro‑
logic function and normal posterior tibial nerve potential [99]. Paek et al. [1] found that prenatal repair of MMC pre‑
vents or reverses hindbrain herniation in the sheep model [1]. Yoshizawa et al. [100] studied fecal incontinence and showed that MMC repair allows for normal development of the anal sphincter [100].
Surgical technique
Aaronson et al. [101] reported that performing intrauter‑
ine MMC repair using a robot‑assisted endoscopic system in the lamb model is technically feasible [101]. Kohl et al.
[102] evaluated the feasibility of a percutaneous fetoscopic approach in sheep and found it to be safe and effective [102].
Aiming at the development of a new endoscopic approach to the correction of MMC, Pedreira et al. [103] assessed the gasless fetoscopy approach. By allowing air to enter the amniotic cavity through cannulas without valve mechanisms (and without gas injection), they created a working space using a uterine lift device and suggested that the gasless technique is an adequate alternative for fetal endoscopic surgery [103, 109].
To assess the feasibility of single‑access fetal endoscopy using intrauterine carbon dioxide as a distension medium in
Fig. 6 Intrauterine surgery for creation of MMC in sheep (left) and postnatal appearance (right)
Table 5 Translational contributions to the study of MMC in the sheep model
Author Year Graft used Surgical approach Main findings Copeland et al.
[96] 1993 Skin graft from mother, Surgicel, and fibrin glue
Fetoscopy Adequate model to study closure of MMC in utero
Meuli‑Simmen
et al. [98] 1995 Latissimus dorsi flap Open Fetal MMC repair may reduce neurological deficit in humans
Paek et al. [1] 2000 Primary repair or
Alloderm Open Alloderm demonstrated retraction of the wound and partial skin growth over the patch; primary repair healed well Kohl et al. [102] 2003 eEPTFE or Tutopatch Fetoscopy, robot‑assisted eEPTFE remains intact and did not adhere to underlying
tissue; tutopatch was completely absorbed Yoshizawa et al.
[100] 2003 Alloderm or Gore‑Tex Open Normal development of rectal muscles and nerves Eggink et al.
[106] Skin closure,
biodegradable matrix, or small intestinal submucosa biomatrix
Open Better neurologic outcome
Sanchez et al.
[107] 2007 Human acellular dermal matrix and biosynthetic cel‑
lulose
Open Biosynthetic cellulose is more adequate
Pedreira et al.
[103] 2008 Biofill or Nexfill Gasless fetoscopy The graft did not adhere and induced the formation of a new fibroblast layer over the exposed medulla in anatomical continuity with the dura mater
Fontecha et al.
[108] 2009 Silastic patch with/
without Marlex mesh, secured with a bioadhesive gel
Open Adequate closure of the neural tube defect, providing regeneration of tissue layers that protect the spinal cord
Pedreira et al.
[109] 2010 Bionext and Integra Gasless fetoscopy Approximation of skin borders in all cases, with visible graft material
Encinas et al.
[110, 111] 2010
2011 Open two‑layer closure, fetoscopic bioglue, or patch
Open, fetoscopy Bioglue allows mild degrees of hydrocephalus and Chiari type II malformation
Fontecha et al.
[112] 2011 Inert patch secured
with surgical sealant Fetoscopy Simple, fast, and satisfactory closure Saadai et al. [113] 2011 biodegradable
nanofibrous scaffolds
Open No inflammatory or fibrotic reaction in fetal tissue
Herrera et al.
[114] 2012 Three‑layer suture (dura mater, muscle, and skin closure) or biosynthetic cellulose patch
Open Synthetic patch preserves nervous tissue and prevents adherence of the spinal cord to the scar
Saadai et al. [115] 2013 Human neural crest stem cells seeded on nanofibrous scaffolds
Open Stem cells survived, integrated, and differentiated into neuronal lineage
Peiro et al. [104] 2013 Silastic patch or MatriDerm secured with Coseal Surgical Sealant
Fetoscopy Feasible and effective
Burgos et al.
[116] 2014 Open 2‑layer closure, open bioglue, or fetoscopic collagen matrix patch
Open, fetoscopy Bladder alterations were completely prevented with open surgery and partially with other coverage modalities
a sheep model, Peiro et al. [104] compared this technique to two‑port fetoscopic coverage and concluded that it is prac‑
tical and effective [104]. Guilbaud et al. [105] assessed the feasibility and the effectiveness of fetoscopic repair with a running single suture using a two‑port access and suggested it may reduce the risk of premature rupture of membranes [105]. Several types of grafts have been tested for the sheep model of MMC, as seen in Table 5.
Stem cell therapy
Fauza et al. [121] introduced the notion of prenatal neural stem cell delivery to the spinal cord as an adjuvant to fetal repair of spina bifida. In addition to the standard MMC cov‑
erage, they delivered murine neural stem cell clones into the spinal cord defect. They found that neural stem cell den‑
sity was the highest within the most damaged areas of the
Table 5 (continued)
Author Year Graft used Surgical approach Main findings Brown et al. [117] 2014 Autologous amniotic
membranes patch or skin closure alone
Open Patch increased protection of spinal cord tissue, but overlying skin failed to close in these fetuses
Watanabe et al.
[118] 2016 Scaffold‑based tissue
engineering Open Enhanced formation of granulation tissue and epithelialization, improved preservation of the spinal cord with less associated damage on histological analysis, and reversal of hindbrain herniation
Mazzone et al.
[119] 2016 Tissue‑engineered
fetal skin In vivo Successful engraftment at 3 weeks
Guilbaud et al.
[120] 2017 Polytetrafluoroethylene patch secured with surgical adhesive
Open, fetoscopy Patch and glue correction may not be the ideal technique to repair fetal MMC
Fig. 7 Surgical procedure to create MMC in mammals
spinal cord, with selective engraftment within those regions, prompting further studies [121].
In order to study the amniotic fluid‑derived stem cells as a potential candidate for application in MMC, Ceccarelli et al. [122] isolated, cultured, and characterized them at the 12th (induction of MMC), 16th (repair of malformation), and 20th week of gestation (delivery). They described the metabolomics and molecular signature variation of stem cells isolated in this model, which may be used as diagnostic tools for in utero identification of neural tube lesions [122].
Wang et al. [123] demonstrated that the use of early ges‑
tation human placenta‑derived mesenchymal stromal cells to enhance in utero repair of MMC resulted in significant and consistent improvement in neurologic function at birth [100]. A year later, they compared the effectiveness of these cells from early‑gestation versus term‑gestation placenta, and their findings suggested that early‑gestation placental stromal cells may exhibit unique properties that further improve the recovery of paralysis following in utero MMC repair [123]. Kabagambe et al. [125] and, later, Vanover et al. [124] investigated the effects of placental mesenchymal stromal cells seeded on porcine small intestine submucosa‑
derived extracellular matrix on hindlimb motor function, and reported improvement of the lamb’s motor function and increased large neuron density [124, 125]. Galganski et al.
[95] determined that in vitro potency assays provide infor‑
mation regarding which placental mesenchymal stromal cell lines produce higher rates of ambulation following in utero treatment of MMC [95].
Conclusions
In summary, surgical animal models of spina bifida have been used for almost 40 years. It is important to notice, how‑
ever, that none of those models truly represents neural tube closing defects and merely represents a defect in the closure
of the neural tube. All models deal with some degree of fetal injury inflicted on an already closed tube and the associated inflammation, as opposed to the true congenital malforma‑
tion. On the same line, after creation of the defect, all models deal with reduced remaining gestation time, as opposed to the long period of exposure after failed closure of the verte‑
bral arches early in pregnancy.
All models involve sedation of the pregnant animal in appropriate gestational age, laparotomy, and uterine incision, followed by some form of fetal exposure method (Figs. 7 and 8). Some authors deliver the fetus up to the thoracic level;
others maintain the fetus totally inside the womb and one of them reported exposing only the tail for better position‑
ing. Some models involve excision of the dura, while others preserve it, and this is an important aspect of the procedure, essentially differentiating a “true” myelomeningocele from other forms of spina bifida.
Future directions, with the use of stem cells and deriva‑
tives in many congenital malformations, are a field open for investigation, and it is not different with MMC. The integration of stem cells with other therapeutic methods, such as seeding the cells in patches and using them to repair the defect might be a topic for future research and decrease sequels in babies born with MMC.
Declarations
Conflict of interest The authors declare that they have no conflict of interest.
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