Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Therapeutic potential of adenovirus-encoding brain-derived neurotrophic factor for spina bifida aperta by intra-amniotic delivery in a rat model

Gene Therapy volume 27, pages 567–578 (2020)Cite this article

Subjects

Abstract

Spina bifida aperta is a type of neural tube defect (NTD). Although prenatal fetal surgery has been an available and effective treatment for it, the neurological functional recovery is still need to be enhanced. Our previous results revealed that deficiencies of sensory, motor, and parasympathetic neurons were primary anomalies that occurred with the spinal malformation. Therefore, we emphasized that nerve regeneration is critical for NTD therapy. We delivered an adenoviral construct containing genes inserted for green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) into the amniotic fluid to investigate its prenatal therapeutic potential for rat fetuses with spina bifida aperta. Using immunofluorescence, TdT-mediated dUTP nick-end labeling staining, and real-time polymerase chain reaction analysis, we assessed cell apoptosis in the defective spinal cord and Brn3a positive neuron survival in the dorsal root ganglion (DRG); a protein array was used to investigate the microenvironmental changes of the amniotic fluid. We found that most of the overexpressed BDNF was present on the lesions of the spina bifida fetuses, the number of apoptosis cells in Ad-GFP-BDNF-transfected spinal cords were reduced, mRNA levels of Bcl2/Bax were upregulated and Casp3 were downregulated compared with the controls, the proportion of Brn3a positive neurons in DRG were increased by activating the BDNF/TrkB/Akt signaling pathway, and most of the significant changes in cytokines in the amniotic fluid were related to the biological processes of regulation of apoptotic process and generation of neurons. These results suggest that intra-amniotic Ad-GFP-BDNF gene delivery might have potential as a supplementary approach to treat congenital malformations of neural tubes.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Characterization of adenovirally expressed GFP and BDNF.
Fig. 2: Improvement of apoptosis in spinal cords of fetuses with spina bifida aperta.
Fig. 3: The percentage of Brn3a+ neurons in DRG increased.
Fig. 4: Amniotic fluid microenvironment changes after Ad-GFP-BDNF injection.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

References

  1. Copp AJ, Adzick NS, Chitty LS, Fletcher JM, Holmbeck GN, Shaw GM. Spina bifida. Nat Rev Dis Primers. 2015;1:15007.

    PubMed  PubMed Central  Google Scholar 

  2. Christianson AL, Howson CP, Modell B. Global report on birth defects: the hidden toll of dying and disabled children. White Plains, NY: March of Dimes Birth Defects Foundation; 2006.

  3. World Health Organization. Global health estimates (GHE)–Cause-specific mortality. 2015. http://www.who.int/healthinfo/global_burden_disease/estimates/en/index1.html. Accessed 14 Apr 2015.

  4. World Health Organization. Global health estimates (GHE)–Disease burden. 2015. http://www.who.int/healthinfo/global_burden_disease/estimates/en/index2.html. Accessed 14 Apr 2015.

  5. Holmes LB, Driscoll SG, Atkins L. Etiologic heterogeneity of neural-tube defects. N Engl J Med. 1976;294:365–9.

    CAS  PubMed  Google Scholar 

  6. Khoury MJ, Erickson JD, James LM. Etiologic heterogeneity of neural tube defects: clues from epidemiology. Am J Epidemiol. 1982;115:538–48.

    CAS  PubMed  Google Scholar 

  7. Mohd-Zin SW, Marwan AI, Abou Chaar MK, Ahmad-Annuar A, Abdul-Aziz NM. Spina bifida: pathogenesis, mechanisms, and genes in mice and humans. Scientifica. 2017;2017:5364827.

    PubMed  PubMed Central  Google Scholar 

  8. Aguiar MJ, Campos AS, Aguiar RA, Lana AM, Magalhaes RL, Babeto LT. Neural tube defects and associated factors in liveborn and stillborn infants. J Pediatr. 2003;79:129–34.

    Google Scholar 

  9. McClain LE, Flake AW. In utero stem cell transplantation and gene therapy: recent progress and the potential for clinical application. Best Pract Res Clin Obstet Gynaecol. 2016;31:88–98.

    PubMed  Google Scholar 

  10. Lorber J. Selective treatment of myelomeningocele: to treat or not to treat? Pediatrics. 1974;53:307–8.

    CAS  PubMed  Google Scholar 

  11. Adzick NS, Sutton LN, Crombleholme TM, Flake AW. Successful fetal surgery for spina bifida. Lancet. 1998;352:1675–6.

    CAS  PubMed  Google Scholar 

  12. Adzick NS, Thom EA, Spong CY, Brock JW 3rd, Burrows PK, Johnson MP, et al. A randomized trial of prenatal versus postnatal repair of myelomeningocele. N Engl J Med. 2011;364:993–1004.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Sacco A, Ushakov F, Thompson D, Peebles D, Pandya P, De Coppi P, et al. Fetal surgery for open spina bifida. Obstet Gynaecol. 2019;21:271–82.

    PubMed  PubMed Central  Google Scholar 

  14. Wei X, Li H, Miao J, Zhou F, Liu B, Wu D, et al. Disturbed apoptosis and cell proliferation in developing neuroepithelium of lumbo-sacral neural tubes in retinoic acid-induced spina bifida aperta in rat. Int J Dev Neurosci. 2012;30:375–81.

    CAS  PubMed  Google Scholar 

  15. Yuan ZW, Lui VC, Tam PK. Deficient motor innervation of the sphincter mechanism in fetal rats with anorectal malformation: a quantitative study by fluorogold retrograde tracing. J Pediatr Surg. 2003;38:1383–8.

    CAS  PubMed  Google Scholar 

  16. Guan K, Li H, Fan Y, Wang W, Yuan Z. Defective development of sensory neurons innervating the levator ani muscle in fetal rats with anorectal malformation. Birth Defects Res A Clin Mol Teratol. 2009;85:583–7.

    CAS  PubMed  Google Scholar 

  17. Jia H, Zhang K, Zhang S, Yuan Z, Bai Y, Wang W. Quantitative analysis of sacral parasympathetic nucleus innervating the rectum in rats with anorectal malformation. J Pediatr Surg. 2007;42:1544–8.

    PubMed  Google Scholar 

  18. Endo M, Zoltick PW, Radu A, Jiang Q, Matsui C, Marinkovich PM, et al. Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa. Gene Ther. 2012;19:561–9.

    CAS  PubMed  Google Scholar 

  19. Muhle C, Neuner A, Park J, Pacho F, Jiang Q, Waddington SN, et al. Evaluation of prenatal intra-amniotic LAMB3 gene delivery in a mouse model of Herlitz disease. Gene Ther. 2006;13:1665–76.

    CAS  PubMed  Google Scholar 

  20. Wu C, Endo M, Yang BH, Radecki MA, Davis PF, Zoltick PW, et al. Intra-amniotic transient transduction of the periderm with a viral vector encoding TGFbeta3 prevents cleft palate in Tgfbeta3−/− mouse embryos. Mol Ther. 2013;21:8–17.

    CAS  PubMed  Google Scholar 

  21. Wan C, Liu NN, Liu LM, Cai N, Chen L. Effect of adenovirus-mediated brain derived neurotrophic factor in early retinal neuropathy of diabetes in rats. Int J Ophthalmol. 2010;3:145–8.

    PubMed  PubMed Central  Google Scholar 

  22. Takano M, Horie H, Iijima Y, Dezawa M, Sawada H, Ishikawa Y. Brain-derived neurotrophic factor enhances neurite regeneration from retinal ganglion cells in aged human retina in vitro. Exp Eye Res. 2002;74:319–23.

    CAS  PubMed  Google Scholar 

  23. Numakawa T, Suzuki S, Kumamaru E, Adachi N, Richards M, Kunugi H. BDNF function and intracellular signaling in neurons. Histol Histopathol. 2010;25:237–58.

    CAS  PubMed  Google Scholar 

  24. Paczkowska E, Luczkowska K, Piecyk K, Roginska D, Pius-Sadowska E, Ustianowski P, et al. The influence of BDNF on human umbilical cord blood stem/progenitor cells: implications for stem cell-based therapy of neurodegenerative disorders. Acta Neurobiol Exp (Wars). 2015;75:172–91.

    Google Scholar 

  25. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer’s disease. Nat Med. 2009;15:331–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Levivier M, Przedborski S, Bencsics C, Kang UJ. Intrastriatal implantation of fibroblasts genetically engineered to produce brain-derived neurotrophic factor prevents degeneration of dopaminergic neurons in a rat model of Parkinson’s disease. J Neurosci. 1995;15:7810–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Bemelmans AP, Horellou P, Pradier L, Brunet I, Colin P, Mallet J. Brain-derived neurotrophic factor-mediated protection of striatal neurons in an excitotoxic rat model of Huntington’s disease, as demonstrated by adenoviral gene transfer. Hum Gene Ther. 1999;10:2987–97.

    CAS  PubMed  Google Scholar 

  28. Kalra S, Genge A, Arnold DL. A prospective, randomized, placebo-controlled evaluation of corticoneuronal response to intrathecal BDNF therapy in ALS using magnetic resonance spectroscopy: feasibility and results. Amyotroph Lateral Scler Other Motor Neuron Disord. 2003;4:22–6.

    CAS  PubMed  Google Scholar 

  29. Makar TK, Bever CT, Singh IS, Royal W, Sahu SN, Sura TP, et al. Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle. J Neuroimmunol. 2009;210:40–51.

    CAS  PubMed  Google Scholar 

  30. Gauthier R, Joly S, Pernet V, Lachapelle P, Di Polo A. Brain-derived neurotrophic factor gene delivery to muller glia preserves structure and function of light-damaged photoreceptors. Invest Ophthalmol Vis Sci. 2005;46:3383–92.

    PubMed  Google Scholar 

  31. Chikar JA, Colesa DJ, Swiderski DL, Di Polo A, Raphael Y, Pfingst BE. Over-expression of BDNF by adenovirus with concurrent electrical stimulation improves cochlear implant thresholds and survival of auditory neurons. Hear Res. 2008;245:24–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Ying X, Tu W, Li S, Wu Q, Chen X, Zhou Y, et al. Hyperbaric oxygen therapy reduces apoptosis and dendritic/synaptic degeneration via the BDNF/TrkB signaling pathways in SCI rats. Life Sci. 2019;229:187–99.

    CAS  PubMed  Google Scholar 

  33. Crowley ST, Fukushima Y, Uchida S, Kataoka K, Itaka K. Enhancement of motor function recovery after spinal cord injury in mice by delivery of brain-derived neurotrophic factor mRNA. Mol Ther Nucleic Acids. 2019;17:465–76.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang L, Lin S, Yi D, Huang Y, Wang C, Jin L, et al. Apoptosis, expression of PAX3 and P53, and caspase signal in fetuses with neural tube defects. Birth Defects Res. 2017;109:1596–604.

    CAS  PubMed  Google Scholar 

  35. Chen C, Zhang J, Sun L, Zhang Y, Gan WB, Tang P, et al. Long-term imaging of dorsal root ganglia in awake behaving mice. Nat Commun. 2019;10:3087.

    PubMed  PubMed Central  Google Scholar 

  36. Lee KS, Zhou W, Scott-McKean JJ, Emmerling KL, Cai GY, Krah DL, et al. Human sensory neurons derived from induced pluripotent stem cells support varicella-zoster virus infection. PLoS ONE. 2012;7:e53010.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Eng SR, Gratwick K, Rhee JM, Fedtsova N, Gan L, Turner EE. Defects in sensory axon growth precede neuronal death in Brn3a-deficient mice. J Neurosci. 2001;21:541–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Wiggins AK, Wei G, Doxakis E, Wong C, Tang AA, Zang K, et al. Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival. J Cell Biol. 2004;167:257–67.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Snider WD. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell. 1994;77:627–38.

    PubMed  Google Scholar 

  40. Huang EJ, Zang K, Schmidt A, Saulys A, Xiang M, Reichardt LF. POU domain factor Brn-3a controls the differentiation and survival of trigeminal neurons by regulating Trk receptor expression. Development. 1999;126:2869–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Lee BD, Yoo JM, Baek SY, Li FY, Sok DE, Kim MR. 3,3’-diindolylmethane promotes BDNF and antioxidant enzyme formation via TrkB/Akt pathway activation for neuroprotection against oxidative stress-induced apoptosis in hippocampal neuronal cells. Antioxidants. 2019;9:E3.

    PubMed  Google Scholar 

  42. Guo W, Ji Y, Wang S, Sun Y, Lu B. Neuronal activity alters BDNF-TrkB signaling kinetics and downstream functions. J Cell Sci. 2014;127:2249–60.

    CAS  PubMed  Google Scholar 

  43. Fasoulakis Z, Theodora M, Tsirkas I, Tsirka T, Kalagasidou S, Inagamova L, et al.  The role of microRNAs identified in the Amniotic Fluid. Microrna. 2019. Epub ahead of print. https://doi.org/10.2174/2211536608666190318105140.

  44. Ferguson MW, Duncan J, Bond J, Bush J, Durani P, So K, et al. Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies. Lancet. 2009;373:1264–74.

    CAS  PubMed  Google Scholar 

  45. Morille M, Van-Thanh T, Garric X, Cayon J, Coudane J, Noel D, et al. New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells. J Control Release. 2013;170:99–110.

    CAS  PubMed  Google Scholar 

  46. Okamura T, Sumitomo S, Morita K, Iwasaki Y, Inoue M, Nakachi S, et al. TGF-beta3-expressing CD4+CD25−LAG3+ regulatory T cells control humoral immune responses. Nat Commun. 2015;6:6329.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Sjors Dahlman A, Blennow K, Zetterberg H, Glise K, Jonsdottir IH. Growth factors and neurotrophins in patients with stress-related exhaustion disorder. Psychoneuroendocrinology. 2019;109:104415.

    PubMed  Google Scholar 

  48. Guaiquil VH, Pan Z, Karagianni N, Fukuoka S, Alegre G, Rosenblatt MI. VEGF-B selectively regenerates injured peripheral neurons and restores sensory and trophic functions. Proc Natl Acad Sci USA. 2014;111:17272–7.

    CAS  PubMed  Google Scholar 

  49. Joyeux L, Danzer E, Limberis MP, Zoltick PW, Radu A, Flake AW, et al. In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice. Hum Gene Ther Methods. 2014;25:197–205.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Danzer E, Schwarz U, Wehrli S, Radu A, Adzick NS, Flake AW. Retinoic acid induced myelomeningocele in fetal rats: characterization by histopathological analysis and magnetic resonance imaging. Exp Neurol. 2005;194:467–75.

    CAS  PubMed  Google Scholar 

  51. Ma W, Wei X, Gu H, Li H, Guan K, Liu D, et al. Sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta. Birth Defects Res A Clin Mol Teratol. 2015;103:772–9.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Key Research and Development Program (2016YFC1000505), the National Natural Science Foundation of China (Grant numbers: 81871219, 81671469, 81901565, 84601292), the Scientific Research Fund of Liaoning Provincial Education Department (LQNK201710).

Author information

Authors and Affiliations

  1. Key Laboratory of Health Ministry for Congenital Malformation, Shengjing Hospital, China Medical University, No. 36, Sanhao Street, Heping District, Shenyang, PR China

    Wei Ma, Xiaowei Wei, Hui Gu, Dan Liu, Wenting Luo, Dong An & Zhengwei Yuan

  2. Department of Pediatrics, The First Affiliated Hospital of China Medical University, Shenyang, PR China

    Dong An

  3. Department of Pediatric Surgery, Shengjing Hospital, China Medical University, Shenyang, PR China

    Yuzuo Bai

Authors
  1. Wei Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaowei Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hui Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wenting Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dong An
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yuzuo Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhengwei Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhengwei Yuan.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ma, W., Wei, X., Gu, H. et al. Therapeutic potential of adenovirus-encoding brain-derived neurotrophic factor for spina bifida aperta by intra-amniotic delivery in a rat model. Gene Ther 27, 567–578 (2020). https://doi.org/10.1038/s41434-020-0131-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-020-0131-2

This article is cited by

Search

Advanced search

Quick links