Histologic and spectroscopic study of pluripotent stem cells after implant in ocular traumatic injuries in a murine model

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Abstract

Abstract Introduction: Ocular trauma is defined as a trauma caused by blunt or penetrating mechanisms on the eyeball and its peripheral structures, causing damage with different degrees of affection with temporary or permanent visual function compromise. Ocular trauma is a major cause of preventable blindness worldwide; it constitutes 7% of all corporal injury and 10% to 15% of all eye diseases. Regenerative medicine research has opened up the possibility to use stem cells as a source of cell replacement, so that experimental studies on embryonic stem cells and bone marrow stem cells have been carried out. In this study, we analyzed the histopathological and spectroscopic changes in ocular tissue with trauma, treated with mouse pluripotent stem cells. Methods: Firstly, mouse embryonic stem cells were seeded. Subsequently, the obtained cells were implanted in a murine model of scleral and retinal damage at the first, second, and fourth weeks post-trauma. At week 12 post-trauma, the eyes were enucleated for histopathologic study (inflammatory response and histological integrity) and spectroscopic analysis by Fourier transform infrared spectroscopy in the attenuated total reflection configuration. Data were analyzed by one-way analysis of variance. Results: Histopathological results showed that the experimental groups treated with stem cells presented a decrease in the inflammatory response, and the histological integrity was restored, which contrasted with the experimental group treated with saline solution. Moreover, in the spectroscopic analysis, characteristic bands of biological samples were observed in all tissues, highlighting in healthy tissues the presence of C=O bond at 1,745 cm-1, which was not observed in the injured and treated tissues. Also, the absorption spectrum of the tissues treated with embryonic stem cells showed bands whose intensity was high at around 1,080 to 1,070 cm-1. It has been reported that these bands are characteristic of pluripotent stem cells. Conclusions: The implant of embryonic stem cells could be a useful therapeutic treatment after traumatic eye injuries or many other eye diseases to reduce the inflammatory response and restore histological integrity. Furthermore, the spectroscopic technique could be used as a complementary technique for detecting stem cell incorporation into various tissues.

Figures

  • Figure 1 Experimental methodology. (A) Experimental groups. To all groups, an ocular trauma (OT) was induced; group 1 received balanced salt solution (BSS), group 2 had an intravitreal implant of 20,000 mouse embryonic stem cells (mESCs) resuspended in 0.025 mL of BSS, and group 3 was treated with an intravitreal implant of 50,000 mESCs resuspended in 0.025 mL of BSS. (B) Experimental methodology. After OT (day 0) and at the first, second, and fourth weeks post-trauma, the groups received their experimental treatment according to the group that they belong to; at 12 weeks post-trauma, rats were sacrificed and the eyes were enucleated for their histopatological and spectroscopy analysis.
  • Figure 2 Stem cell culture. Mouse embryonic stem cells (400×) integrating a colony that is under a mouse embryonic fibroblast monolayer.
  • Figure 4 Scleral inflammatory response and histological integrity. Scleral tissues (400×). (A) In group 1, moderate inflammatory response characterized by the presence of lymphocytes (↑) and loss of the arrangement of the collagen fibers are shown. (B) In group 2, few lymphocytes (↑) and a slight alteration in the histological integrity were observed. (C) In group 3, cells involved in the inflammatory response are not shown, and the arrangement of the collagen fibers was restored, which means that the scleral histology was recovered.
  • Figure 3 Histology of the ocular trauma in a murine model. Alteration of the normal histological conformation of the retina characterized by dissemination of the retinal neuron nucleuses 48 hours after ocular trauma (400×).
  • Figure 5 Inflammatory response. The inflammatory response was graded cell infiltrate. None of the experimental groups showed any signs of severe had a moderate alteration, whereas most members of groups 2 and 3 (gro inflammatory response (P <0.0001).
  • Figure 6 Retinal histological integrity. Retinal tissues (100×). (A) In group 1, histological integrity, characterized by dissemination of neuronal cell nuclei, was not retained. (B) In group 2, restored architecture is observed with homogenization of the retinal layers. (C) In group 3, restored architecture with normal neuronal cellularity is shown.
  • Figure 7 Histological integrity. The histological integrity was graded as restored, mild alteration, moderate alteration, and severe alteration, according to the presence of fibroblastic reaction, loss of the longitudinal axis of the fibroblasts or fibrocytes, arrangement of the collagen fibers, displacement of normal tissue structures, and recapilarization. None of the experimental groups showed any signs of severe alterations of histological integrity. Most members of group 1 (control group) had a moderate alteration, whereas groups 2 and 3 (groups that received mouse embryonic stem cells) showed a restored histological integrity (P <0.0001).
  • Figure 8 Infrared absorption spectrum. Fourier transform infrared spect representative histological sections in three different conditions: healthy tis embryonic stem cells. a.u., arbitrary units.

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CITATION STYLE

APA

Vázquez-Zapién, G. J., Rojas-López, M., Delgado-Macuil, R. J., Martínez-Nava, L. R., Pérez-Ishiwara, D. G., & Mata-Miranda, M. M. (2014). Histologic and spectroscopic study of pluripotent stem cells after implant in ocular traumatic injuries in a murine model. Stem Cell Research and Therapy, 5(6). https://doi.org/10.1186/scrt509

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