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Journal of Pediatric Surgery (2013) 48, 2446–2452
Morphology of nervous lesion in the spinal cord and bladderof fetal rats with myelomeningocele at differentgestational ageJian Shena, Guomin Zhoub, Hong Chen a, Yunli Bi c,⁎
aChildren's Hospital of Fudan University, Shanghai, PR ChinabDepartment of Anatomy, Histology and Embryology, Shanghai Medical College of Fudan University, Shanghai, PR ChinacDepartment of Urology, Children's Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, PR China
Received 21 August 2013; accepted 26 August 2013
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Key words:Neurogenic bladderdysfunction;Myelomeningocele;Fetus;Nevous lesion
AbstractObjective: To analyze the development and innervation of bladder smooth muscle and lesions of thespinal cord in fetal rats with meningomyelocele (MMC) at different gestational ages and to investigateinteractions between spinal cord lesions and bladder.Method: Each fetus was assigned to the MMC group or the normal group. Each group was furtherdivided into three subgroups by gestational age: E16, E18, and E20 (embryonic days 16, 18, and 20,respectively). α-Actin and neurotubulin-β-III were analyzed in the bladder, and GFAP and VAChTwere analyzed in the lumbosacral spinal cord by immunohistochemistry. Photographs were taken todetermine the integrated optical density of each sample.Results: Neurotubulin-β-III was significantly lower in the MMC group than in the normal group at allfetal ages. Abundant α-actin was detected in both groups at all fetal ages. No significant difference wasfound between the MMC group and the normal group at any fetal age. At E16 and E18, no GFAP-positive astrocyte was detected in the MMC group or the normal group. At E20, numerous GFAP-positive astrocytes were detected in the MMC group, with significant difference from the normalgroup. VAChT was detected less in the MMC group than in the normal group at all fetal ages withsignificant differences.Conclusion: Bladder smooth muscle of fetal MMC rat seems morphologically normal in development,while the innervation of the bladder smooth muscle is markedly decreased centrally and peripherally.Astrocytosis appears at a later embryonic stage, which could be a concern in the nerve repair of thespinal cord.© 2013 Elsevier Inc. All rights reserved.
⁎ Corresponding author. Tel.: +86 13162008989.E-mail addresses: [email protected] (J. Shen),
[email protected] (G. Zhou), [email protected]. Chen), [email protected] (Y. Bi).
022-3468/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.jpedsurg.2013.08.021
Myelomeningocele (MMC) is the most common cause ofneurogenic bladder dysfunction (NBD) in children. MMCalso can lead to paraplegia, incontinence, hydrocephalus,sexual dysfunction, skeletal malformation, and mentalimpairment. MCC, especially in embryonic development,
2447Nervous lesion in fetal rats with myelomeningocele
is studied less frequently than is spinal cord injury, which isthe most common cause of NBD in adults. The conclusionsof previously published literature about embryonic bladderdevelopment of MMC fetus remain controversial. We used anew fetal MMC rat model to observe the morphology ofneural distribution and lesions in the spinal cord and bladderat different gestational ages.
1. Materials and methods
1.1. Animal preparation and retinoic acid exposure
Time-dated primigravida Sprague–Dawley rats weighing220 to 320 g (Animal Laboratory of Fudan University,Shanghai), were used for this study. All-trans retinoic acid(RA; Sigma-Aldrich Chemical, Shanghai) was dissolved atroom temperature in olive oil. The RA solution was usedwithin 1 h of preparation. After a brief exposure toisoflurane, pregnant rats were gavage fed 60 mg/kg of RAdissolved in 2 ml of olive oil at embryonic day 10 (E10).Control rats were fed 2 ml of olive oil alone. A total of 39pregnant rats were included in this study: 30 were exposed toRA and 9 were exposed to olive oil alone.
1.2. Grouping and collection of fetuses
Pregnant rats were assigned to the RA-exposed group orto the control group. Both groups were divided into threesubgroups according to the time of collection of fetuses: E16,E18, or E20 (embryonic days 16, 18, and 20, respectively).
Pregnant rats were anesthetized with isoflurane. Fetuseswere collected by cesarean section at gestational ages E16,E18, and E20. Subsequently, dams were euthanized bycervical dislocation. There were 10 pregnant rats in eachRA-exposed subgroup and 3 pregnant rats in each controlsubgroup.
Randomly selected MMC fetuses from pregnant rats ofthe RA-exposed group were defined as the MMC group(M group), and normal fetuses from pregnant rats of controlgroup were defined as the normal group (N group). Boththe MMC group and the normal group were divided intothree subgroups: E16, E18, and E20. Each subgroup hadfive fetal rats. Each fetus was perfused with 4%paraformaldehyde 0.1 M PBS via the heart, and their spinalcolumns and bladders were dissected in PBS under adissecting microscope.
1.3. General morphology and immunohistochemistry
Lumbosacral spinal columns and bladders were dehy-drated and embedded in paraffin, and then were sequentiallysectioned at 5-μm thickness.
For general morphology, sections were deparaffinized,rehydrated with distilled water, and stained with hematoxylin
and eosin according to standard protocols. Sections forimmunohistochemistry were air-dried overnight in a 56 °Cincubator, deparaffinized, and rehydrated with distilledwater. The sections were then immersed in sodium citratebuffer solution, and were incubated in a water bath at 97 °Cfor 8 min, washed in 0.01 M PBS, transferred to 0.01 MPBS containing 10% goat serum and 0.1% Triton X-100 toblock endogenous peroxidase, and incubated at 37 °C for30 min.
Glial fibrillary acidic protein (GFAP) and vesicularacetylcholine transporter (VAChT) were used for antigendetection in the spinal cord, and α-smooth muscle actin (α-SMA) and neurotubulin-β-III were used for antigendetection in the bladder. The following primary antibodieswere used in this study: anti-α-SMA (Santa Cruz, USA),anti-neurotubulin-β-III (Millipore, USA), anti-GFAP (Milli-pore, USA), and anti-VAChT (Santa Cruz, USA). Chem-MateTM EnVisionTM detection kit, Peroxidase/DAB, Rabit/Mouse (DAKO, USA) was used as secondary antibody andchromogenic agent according to standard protocols.
1.4. Data collection and statistical analysis
Photomicrographs of all the sections were taken usingLeica QWin imaging software (Leica, Germany). Integratedoptical density of the concerned region in the immunohis-tochemical sections was measured by Image-Pro Plus 5.1.Significance was defined as P ≤ 0.05. All analyses wereperformed by SPSS 16.0.
2. Results
One hundred twenty-nine (45.10%) of 286 RA-exposedfetuses developed MMC. In the control group, no MMC wasdetected. The RA-exposed fetal rats developed malforma-tions in addition to myelomeningocele, including malforma-tions of the lower limbs or anus, as well as encephalocele(Fig. 1). Fetal rats with coexisting myelomeningocele andencephalocele were excluded from this study.
2.1. HE staining
The gray matter and white matter of spinal cords in thenormal group were clearly delineated. The vertebraedeveloped well without spina bifida (Fig. 2-a). Preganglionicautonomic neurons with hyperchromatic nuclei weredetected in the dorsal lateral nucleus in the ventral hornand in the sacral parasympathetic nucleus in the intermedio-lateral cell group (arrow in Fig. 2-a). Defects of the vertebralarch and spina bifida were found in MMC fetuses. Defects inthe spinal cords of MMC fetuses varied from mild deformityto total eversion or split. The structure of the gray and whitematter was disorganized, and vascular proliferation andhyperemia were detected (Fig. 2-B, b).
Fig. 1 Various malformations after RA exposure. (A) MMC with cephalocele. (B) Synpodia. (C) Mild MMC. (D) Gigantic MMC withcaudal regression syndrome.
ig. 2 Sections of lumbosacral spinal cord by HE stained at E20.) Full view of normal spinal cord (10 × 5). (a) Magnified view ofe frame in panel A showing the darkly stained neurons (red arrow,0 × 40). (B) Full view of MMC spinal cord (10 × 10). (b)agnified view of the frame in panel B showing vascularroliferation in the MMC spinal cord (10 × 40).
2448 J. Shen et al.
2.2. IHC staining
2.2.1. Neurotubulin-β-IIIAt E16, neurotubulin-β-III was detected in fetal bladder
muscle in both the MMC group and the normal group, andwas quite sparse in MMC fetuses. At E18 and E20,neurotubulin-β-III increased in both groups, and wasrelatively less in the MMC group (Fig. 3). Integrated opticaldensity of neurotubulin-β-III was significantly less in theMMC group than in the normal group at E16, E18, and E20(Fig. 4). There were statistical differences, and P values were0.047, 0.033, and 0.008 respectively.
2.2.2. α-Smooth muscle actin (α-SMA)Abundant α-SMA was detected in the fetal bladder
muscle in both the MMC group and the normal group at E16,E18 and E20 (Fig. 3). Differences of integrated opticaldensity of α-SMA between the MMC group and the normalgroup at E16, E18, and E20 were not significant (Fig. 4).
2.2.3. GFAPAt E16 and E18, there was little GFAP expression in the
spinal cord of either group. At E20, there was little GFAP-positive stain in the normal group, while there were abundantGFAP-positive astrocytes in the dorsal region of the spinalcord in the MMC group (Fig. 3). Differences of integratedoptical density of GFAP between the MMC group and thenormal group at E16 and E18 were not significant, whilethere was a significant difference at E20, with a P value of0.034 (Fig. 4).
2.2.4. VAChTThere was abundant VAChT expression in the dorsal
lateral nucleus of the spinal cord in the normal group at allfetal ages, but less VAChT expression in the MMC group(Fig. 3). Integrated optical density of VAChT was signifi-
F(Ath1Mp
Fig. 3 Representative photographs of normal group (N) and MMC group (M) sections at different gestational age afterimmunohistochemical staining (10 × 40).
2449Nervous lesion in fetal rats with myelomeningocele
cantly less in the MMC group than in the normal group atE16, E18, and E20 (Fig. 4). There were statistical differences,and P values were 0.027, 0.039, and 0.012, respectively.
3. Discussion
In early-stage NBD, there are many types of bladderdysfunction. In advanced-stage NBD, detrusor–sphincterdiscoordination and high pressure in the bladder causebladder hypertrophy, fibroplasia, vesico-ureteral reflux,dilation of the upper urinary tract, and renal compromise.In the literature there are few studies of the relationshipbetween lesions of the spinal cord and the bladder.
3.1. Neurotubulin-β-III and neurons in bladdermuscular layer
Neurotubulin-β-III is a specific marker of neurons that iswidely used in neurobiological research. In a study using thesame animal model, neurotubulin-β-III was first detected at18 days of gestation. While an increase of this neurotubu-lin-β-III-positive nerve was observed with increasinggestational age within the circular and longitudinal musclelayers in the bladders of normal fetal rats, the density ofneurotubulin-β-III-positive nerve fibers within the detrusormuscle of MMC bladders was markedly reduced at E20, and
almost completely absent at term [1]. Similarly in our study,neurotubulin-β-III-positive nerve fibers were significantlyreduced within the muscle layer of the MMC fetal bladderthroughout gestation.
Neural density was also found to be reduced in the smoothmuscle layer of the MMC fetal bladder in a study of humanfetuses [2]. In pediatric myelodysplastic bladders, muscariniccholinergic receptors were found to be significantly reducedcompared with those in normal pediatric bladders [3].
3.2. α-SMA and bladder smooth muscle
α-Smooth muscle actin is a specific marker protein ofsmooth muscle. We found that there was no significantdifference in the expression of α-SMA in MMC bladdersversus normal bladders throughout gestation.
Many studies have shown that integrated innervation wasextremely important to the development of the urogenitalsystem [4,5], while in our MMC fetal rat model, it appearedthat reduction of innervation did not have a morphologicaleffect on smooth muscle in the fetal bladder. Danzer et al. [1]showed a similar finding in the same rat model, while in astudy of human fetuses, Shapiro et al. [2] found that bladdersmooth muscle was less well differentiated in those withMMC. In the bladder and bladder neck of those withmyelomeningocele, smooth muscle was sparse, with anexcess of collagen in an interfascicular and intrafasciculardistribution. Other studies showed that the bladder function
2450 J. Shen et al.
of children with myelodysplasia deteriorated with time [6,7].Although discordant results in the above studies may beconfusing, they may be explained by the fact that a rat fetus atterm (E22) corresponds developmentally to an early-second-trimester human fetus [1], and in that period, such changes inbladder smooth muscle may not yet have occurred. Despitethe development of normal morphology of smooth muscle infetal rats with MMC, the contractility responses of MMCmuscle strips are significantly reduced [1].
3.3. GFAP and spinal cord lesions
Glial fibrillary acidic protein is a kind of specificintermediate filament protein of the astrocyte, and is oftenused as a marker of astrocytes. After a nerve is injured,astrocytes will fill the defect and form the glial scar. Thus,reactive astrocytes are considered to be an early and sensitivemarker of neurotoxicity [8]. In animal-model studies of
Fig. 4 Integrated optical density of normal group (N) and MMC groupstaining.
spinal cord injury (SCI), proliferation of GFAP-positiveastrocytes could always be detected after SCI [9,10]. But inthe fetus, this process may be different. Oyanagi et al. [11]found that astrogliosis and glial scar were much lessextensive in fetal rats than that in adult rats after the samebrain injury. Fujimoto et al. [12] found that GFAP expressionwas only slightly increased 1 week after transection of thespinal cord of fetal rats, and decreased to normal levels5 weeks after injury. No glial scars were observed. Incontrast, GFAP expression increased continuously from3 days to 5 weeks after the same injuries in adult rats, andapparent glial or connective tissue scars were observed.
Our study found that at E16 and E18, there was littleGFAP-positive stain in the spinal cord of either group. AtE20, there was little GFAP-positive stain in the normalgroup, while GFAP was strongly expressed in the MMCgroup, especially in the dorsal region, where the spinal cordwas most severely affected. Numerous GFAP-positive
(M) sections at different gestational age after immunohistochemical
2451Nervous lesion in fetal rats with myelomeningocele
astrocytes were detected in the dorsal region in a high-powermicroscopic field. Similar results were found in a ct/lp mouseMMC model, also by immunohistochemical methods. Aprogressive increase in astrocytes in the spinal cord of allmouse fetuses was found between days 14.5 and 18.5 ofgestation. This increase was significantly higher in theplacodes of mice with MMC than in those of normal mice,particularly in the posterior region [13]. In a study ofamniotic fluid GFAP levels using the same rat model wehave used in our study, amniotic fluid GFAP levels werefound to be similar in both groups at E14, 16, and 18,respectively. Compared with control fetuses, amniotic fluidGFAP levels were significantly increased in MMC fetuses atE20 and 22 [14].
When the central nervous system is disrupted, astrocytesare activated and proliferate to become reactive astrocyteswith large cell processes and rich soma. These cellssynthesize neurotrophic factors and growth factors thatplay a large role in nerve repair; however, in mature centralnervous tissue, reactive astrocytes inhibit repair by forming aglial scar at the injury site [12]. Proliferating reactiveastrocytes are critical to scar formation and function toreduce the spread and persistence of inflammatory cells, tomaintain the repair of the blood–brain barrier (BBB), todecrease tissue damage and lesion size, and to decreaseneuronal loss and demyelination [15,16].
3.4. VAChT and innervation
The smooth muscle forming the detrusor muscle ofbladder is innervated via the pelvic nerve by parasympatheticpreganglionic motoneurons in the intermediolateral cellgroup or sacral parasympathetic nucleus. The rat sacralparasympathetic nucleus is located in the L6–S1 spinalsegments [17]. The pudendal nerve innervates the intrinsicexternal urethral sphincter, which forms part of the pelvicfloor musculature. In most vertebrates, the pelvic floormotoneurons are located in the so-called nucleus of Onuf(ON). In the rat, ON consists of two separate nuclei [18,19].The dorsolateral nucleus contains motoneurons innervatingthe ischiocavernosus muscle and the external urethralsphincter [20].
Vesicular acetylcholine transporter (VAChT) is a vesiclemembrane protein that is responsible for the uptake ofacetylcholine into synaptic vesicles [21]. The most importantfeature of VAChT is its fairly restricted localization oncholinergic synaptic vesicles, and therefore, every choliner-gic terminal including autonomic and motor termini can beclearly visualized by immunohistochemistry using anti-VAChT antibody [22,23].
In a spinal-cord-injury rat model, 3–15 days after SCI,VAChT-positive nerve terminals around the motor neuronsin the ventral horn and pelvic ganglia were markedlydecreased. In addition the VAChT-positive terminals in theintermediolateral nucleus and sacral parasympathetic nucle-
us were dramatically decreased [24]. The expression ofVAChT in MMC has not been reported by far. In our study,VAChT was found to be markedly decreased in theintermediolateral cell group or in the sacral parasympatheticnucleus in the MMC group throughout the gestation, whichmeans that the parasympathetic preganglionic motoneuronsinnervating the detrusor muscle were markedly decreased. Itwas consistent with the decease of neurotubulin-β-III inbladder smooth muscle.
A major limitation of this study is relatively shortgestation of rat model and nonsurvivability of the pupsafter birth. So it will be almost impossible to observe thedevelopment and innervation of bladder smooth muscle afterbirth. And further studies are needed to reveal whyastrocytosis appear in the late embryonic stage and whatrole do the astrocytes play in the spinal cord lesions ofmyelomeningocele rats.
4. Conclusion
Smooth muscle of the bladder in fetal rats withmyelomeningocele is morphologically normal, while theinnervation of the smooth muscle of the bladder is markedlydecreased centrally and peripherally. Astrocytosis appears ina later embryonic stage, which could be related to nerverepair in the spinal cord.
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