Compromised Neurotrophic and Angiogenic Regenerative Capability during Tendon Healing in a Rat Model of Type-II Diabetes

Abstract

Metabolic diseases such as diabetes mellitus type-II (DM-II) may increase the risk of suffering painful connective tissue disorders and tendon ruptures. The pathomechanisms, however, by which diabetes adversely affects connective tissue matrix metabolism and regeneration, still need better definition. Our aim was to study the effect of DM-II on expressional changes of neuro- and angiotrophic mediators and receptors in intact and healing Achilles tendon. The right Achilles tendon was transected in 5 male DM-II Goto-Kakizaki (GK) and 4 age-matched Wistar control rats. The left Achilles tendons were left intact. At week 2 post-injury, NGF, BDNF, TSP, and receptors TrkA, TrkB and Nk1 gene expression was studied by quantitative RT-PCR (qRT-PCR) and their protein distribution by immunohistochemistry in intact and injured tendons. The expression of tendon-related markers, Scleraxis (SCX) and Tenomodulin (TNMD), was evaluated by qRT-PCR in intact and injured tendons. Injured tendons of diabetic GK rats exhibited significantly down-regulated Ngf and Tsp1 mRNA and corresponding protein levels, and down-regulated Trka gene expression compared to injured Wistar controls. Intact tendons of DM-II GK rats displayed reduced mRNA levels for Ngf, Tsp1 and Trkb compared to corresponding intact non-diabetic tendons. Up-regulated Scx and Tnmd gene expression was observed in injured tendons of normal and diabetic GK rats compared to intact Wistar controls. However, these molecules were not up-regulated in injured DM-II GK rats compared to their corresponding controls. Our results suggest that DM-II has detrimental effects on neuro- and angiotrophic pathways, and such effects may reflect the compromised repair seen in diabetic Achilles tendon. Thus, novel approaches for regeneration of injured, including tendinopathic, and surgically repaired diabetic tendons may include therapeutic molecular modulation of neurotrophic pathways such as NGF and its receptors.

Fig 1. Relative expression of Tnmd and Scx mRNA in Achilles tendons.

(a Scx and (b Tnmd gene expression in intact and ruptured Achilles tendons of Wistar and diabetic GK rats at day 14 post-rupture. Results are expressed as mean ± SEM with n = 4 in Wistar groups and n = 4 in diabetic GK groups (ANOVA followed by Fisher’s protected least significant difference test).

Fig 2. Relative expression of Ngf, Bdnf, Tsp1, Nk1, Trka and Trkb mRNA in the Achilles tendons.

a) Relative gene expression of Ngf b) Bdnf, c) Tsp1, d) Nk1, e) Trka and f) Trkb in the intact and ruptured Achilles tendons of Wistar and diabetic GK rats at day 14 post-rupture. Results are expressed as mean ± SEM with n = 4 in Wistar groups and n = 5 in diabetic GK groups. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 compared to intact and ruptures Wistar and ^#P ≤ 0.05 compared to intact GK group (ANOVA followed by Fisher’s protected least significant difference test).

Fig 3. NGF, BDNF and TSP1 expression in Achilles tendon.

Expression of NGF, BDNF and TSP1 in intact and ruptured tendon of Wistar and diabetic GK rats at day 14 post-rupture. Original magnification is 20x; bars = 100 μm. (I; Intact part of the tendon and R; Ruptured area).

Fig 4. NK1 and Trk expression in Achilles tendon.

Expression of NK1 and Trk in intact and ruptured tendon of Wistar and diabetic GK rats at day 14 post-rupture. Original magnification is 20x; bars = 100 μm. (I; Intact part of the tendon and R; Ruptured area).