Experimental diabetes induces structural, inflammatory and vascular changes of Achilles tendons

Abstract

This study aims to demonstrate how the state of chronic hyperglycemia from experimental Diabetes Mellitus can influence the homeostatic imbalance of tendons and, consequently, lead to the characteristics of tendinopathy. Twenty animals were randomly divided into two experimental groups: control group, consisting of healthy rats and diabetic group constituted by rats induced to Diabetes Mellitus I. After twenty-four days of the induction of Diabetes type I, the Achilles tendon were removed for morphological evaluation, cellularity, number and cross-sectional area of blood vessel, immunohistochemistry for Collagen type I, VEGF and NF-κB nuclear localization sequence (NLS) and nitrate and nitrite level. The Achilles tendon thickness (µm/100g) of diabetic animals was significantly increased and, similarly, an increase was observed in the density of fibrocytes and mast cells in the tendons of the diabetic group. The average number of blood vessels per field, in peritendinous tissue, was statistically higher in the diabetic group 3.39 (2.98) vessels/field when compared to the control group 0.89 (1.68) vessels/field p = 0.001 and in the intratendinous region, it was observed that blood vessels were extremely rare in the control group 0.035 (0.18) vessels/field and were often present in the tendons of the diabetic group 0.89 (0.99) vessels/field. The immunohistochemistry analysis identified higher density of type 1 collagen and increased expression of VEGF as well as increased immunostaining for NFκB p50 NLS in the nucleus in Achilles tendon of the diabetic group when compared to the control group. Higher levels of nitrite/nitrate were observed in the experimental group induced to diabetes. We conclude that experimental DM induces notable structural, inflammatory and vascular changes in the Achilles tendon which are compatible with the process of chronic tendinopathy.

Figure 1. Glycemic Expression ml/dL at three evaluations.

CG – Control group and DG – Diabetic group. *- p = 0.001. Student t test to determine statistical differences of CG and DG.

Figure 2. Thickness of the Achilles tendons in the Control Group – CG and the Diabetic Group – DG with results are exhibited in terms of thickness value/100 g of the animal's weight.

A. Quantification of cell density – Fibrocytes, fibroblast and Total Cells in the Control Group – CG and the Diabetic Group – B. H.E sections (10 most proximal X400 viewing fields).The values are expressed as means and standard errors. * – p<0.05 Student-t Test for independent samples showing statistical differences between the groups studied.

Figure 3. Density and Cross Sectional Area of blood vessels in the Achilles tendon in the Control Group – CG and the Diabetic Group – DG in the intratendinous and peritendinous regions.

The values are expressed as means and Standard Deviation of the Mean (SEM). * –p<0.05 Student-t Test for independent samples between the groups studied. Normal tendon –A; Tendon of the diabetic group with arrows pointing to blood vessels –B. 400X – HE.

Figure 4. Density of mast cells in the Achilles tendon in the Control Group – CG and the Diabetic Group – DG.

Normal tendon. A; Tendon of the diabetic group with arrows pointing to mast cells –B. * –p<0.05 Student-t Test for independent samples between the groups studied –400X – Toluidine blue.

Figure 5. Level of nitric oxide product – nitrite/nitrate (NOx) in the Achilles tendon in the Control Group – CG and the Diabetic Group – DG.

* – p<0.05 Student-t Test for independent samples between the groups studied.

Figure 6. Immunomarking the density of Type I Collagen and VEGF. Density of type I Collagen in the Achilles tendon in the Control Group.

A; Increased of the density of type I Collagen in the Achilles tendon in the Diabetic Group and disorganization in the Extracellular Matrix –B; The absence of VEGF expression in the control group –D; Expression of VEGF in the diabetic group –E; Negative Controls –C and F. 400X. Relative stained area was quantified using National Institutes of Health ImageJ software. The bar graph summarizes average values of each group for type 1 collagen –G and VEGF –H. The values are expressed as means and Standard Deviation of the Mean (SEM). * –p<0.05 Student-T Test for independent samples showing statistical differences between the groups studied.

Figure 7. A greater degree of Immunomarking of NF-κB nuclear expression was observed in the diabetic group.

B; when compared to the control group – A. Highlights of immunomarking of NF-κB nuclear expression in the diabetic group – C. *-p = 0,001 Student-t Test for independent samples between the groups studied.

Figure 8. Signaling pathways in diabetic tendinopathy.

Initially, the state of chronic hyperglycemia results in the activation of NF-κB, which leads to the increased expression of target genes such as VEGF and NOSi. These, in turn, result in increased NO and vascularization. This increased vascularization associated with cell proliferation and possible migration causes hypercellularity and the rise of disorganized deposition of type 1 collagen. Mast cells increased as a result of an inflammatory process of the tendon denoted by the increase of nitrite and nitrate indicative of increased NO also contribute to the increase of VEGF and therefore to increased vascularity.