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CHARACTERISTICS OF NEUROPEPTIDE-CONTAINING INNERVATION, TISSUE REMODELLING, GROWTH AND VASCULARITY IN THE NASAL TISSUE OF THE CLEFT LIP PATIENTS 23045 Evija Balode, Dr. habil. med. prof. Māra Pilmane Rīga Stradiņš University, Institute of Anatomy and Anthropology; Department of Morphology Introduction Results The pathogenesis of non-syndromic cleft and lip palate (CLP) development is a complex, yet not fully understood mechanism. There have been few studies looking at the nasal tissue in the aspect of CLP, however none, have looked at innervation, tissue remodelling, growth and vascularity altogether. CLP affected tissue revealed dense and loose connective tissue, adipose cells and hyaline cartilage, along with numerous CD34 positive endotheliocytes (Fig. 1A, Table 1) and VEGF positive neoangiogenesis regions (Fig. 1B, Table 1). We observed moderate to numerous PGP 9.5 positive nerve fibres (Fig. 1C, Table 1). Number of positive cells was moderate to numerous for TGFβ1, MMP9 and TIMP2 and few to moderate for MMP2 (Fig. 2, Table 1). Aim of the study Our aim was to detect the appearance and distribution of factors regulating the tissue remodelling, innervation, growth and vascularity in nasal tissue affected by complete cleft lip and palate. Fig. 2. (A) Moderate number of TGFβ1 positive structures in the nasal connective tissue. TGFβ1 IMH, X200. (B) Note the numerous MMP2 positive chondrocytes and moderate number of positive cells in connective tissue cells in the nasal tissue affected by CLP. MMP2 IMH X200. (C) Numerous MMP9 positive chondrocytes and moderate to numerous number of positive cells in connective tissue observed in CLP affected nose. MMP9 IMH, X200. (D) Numerous TIMP2 positive structures in the connective tissue of the cleft nose. TIMP2 IMH, X200. Materials and methods The study included 17 surgery materials from 15 patients and 7 control tissue samples taken from the apex nasi region. Biotin-streptavidin immunohistochemistry was performed for PGP 9.5, TGFβ1, VEGF, CD34, MMP2, MMP9 and TIMP2. The results were evaluated semiquantitatively. For statistical analysis, we used Mann-Whitney U test in SPSS software, version 22.0 (IBM Company, Chicago, USA). A B C D Table 1. The average results of semiquantitative evaluation of PGP 9.5, TGFβ1, CD34, VEGF, MMP2, MMP9 and TIMP2 expression in the tissue of CLP patients and control tissue, and results of Mann-Whitney U test. Marker PGP 9.5 TGFβ1 CD34 VEGF MMP2 MMP9 TIMP2 CLP ++/+++ +++ +/++ Control ++ p 0.006 0.109 0.203 0.001 0.002 0.013 <0.001 Fig. 1. (A) Hyaline cartilage of intranasal septa invaded by connective tissue with CD34 positive endotheliocytes in the blood vessels. CD34 IMH, x200. (B) Note the numerous VEGF positive chondrocytes and a moderate amount of VEGF positive endothelial cells in the blood vessel of the nasal tissue affected by CLP. VEGF IMH, x200. (C) Numerous PGP 9.5 positive nerve fibres in the nasal blood vessels. PGP 9.5 IMH, x200. Conclusions The moderate expression of TGFβ1 demonstrates an active ECM remodelling, carried out by proteinases. An increased number of VEGF, MMP2, MMP9, and TIMP2 demonstrates balanced tissue remodelling and neoangiogenesis of the CLP nasal tissue. Hyaline cartilage of intranasal septum is an example of balance between tissue degradation and its suppression, demonstrated by a relationship between MMPs and TIMPs and by the presence of VEGF. Increased innervation combined with persistent TGFβ expression and tissue ischemia, might be a sign of previous inflammation. A B C
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