Células tronco mesenquimais autólogas infundidas por perfusão regional venosa na terapia da laminite crônica em equinos

Abstract

Laminitis stands out among the affections in locomotive limbs as the main cause of the removal of horses from their activities. A little studied therapeutic possibility are the mesenchymal stem cells. The objectives of this study were to evaluate blood perfusion, hoof growth rate and spatial relationship between the hoof capsule and the distal phalanx of horses with chronic laminitis and who were treated with adCTM infused by regional perfusion in the digital vein in the thoracic limb comparing with the contralateral limb infused with placebo solution, describe the histopathological findings of horses with chronic laminitis and classify the degree of injury pre- and post-treatment with adCTM or placebo; to evaluate epidermal lamellar histomorphometry before and after treatment with adCTM or placebo; to test the immunohistochemical protocol for the p63 antibody (progenitor epithelial cells) in lamellar samples of healthy horses with chronic laminitis. Additionally, a possible systemic effect of adCTM was observed comparing the results obtained from the infused member with those of the contralateral limb (placebo). The experimental methodology was approved by the Ethics Committee (CEUA / UFMG 282/2016). Six equines, Mangalarga Marchador, females, weighing between 381 ± 11 kg, with mean age of 8.3 ± 4.7 years, body score 6 ± 1 (score 1 to 9), attended at HV-UFMG, and identified as having chronic laminitis through history, confirmed by physical and radiographic examination, all of them being classified as grade 1 to 2 of claudication Obel. The study involved the evaluation in two equivalent periods of 30 days, before treatment and after treatment. Each horse received three treatments with adCTM [2 x 107 cells / suspended in 3 ml of PBS and completed to the volume of 20 ml with 0.9% NaCl (SF) - adCTM group] or placebo (SF - placebo group) in the same volume infused by regional perfusion of lateral digital vein for 30 minutes, repeated every 15 days. To verify the blood perfusion of the hoof, digital venographs were performed on both thoracic limbs. Each projection was evaluated for the vascular filling level at pre-determined, pre and posttreatment sites. As a method of evaluating the growth rate of the hoof capsule, the metal spheres were implanted in the wall of the hoof, accompanied by radiographic examinations. For growth monitoring before the application of any treatment, two radiographic exams were performed with a thirty-day interval (A1 and A30). In order to verify the full effect of the adCTM, two moments were also considered with a thirty-day interval, one fifteen days after the third application of adCTM (D30) and another thirty days before this final moment (D1). The evaluation of the spatial relationship between the distal phalanx and the hoof capsule was obtained by computer program. Samples of lamellar biopsy were stained with hematoxylin-eosin and periodic acid from Schiff, and were classified according to degree of histological lesion, epidermal lamellar histomorphometry and tested for an immunohistochemical protocol for p63 and analyzed by pre and post-treatment microscopy. Considering the sum of the venography scores, there was a significant improvement in the adCTM group 15 days after the third application when compared to the pretreatment test (P <0.03). This difference was also manifested specifically in the variables perfusion of lamellar dorsal vessels (LDV, P <0.03) and circumflex-lamellar junction (CLJ, P <0.03). The adCTM group showed a greater distance between the coronary border and the insertion point of the beads at the end of treatments (D30) compared to the placebo group (P <0.03). On the spatial relationship between the distal phalanx and the hoof capsule, positive effects were also observed in the limbs treated with adCTM, promoting stability of the distal phalanx compared to the placebo group. The results of analyzes of the degree of global histological lesion in pre and post-treatment lamellar tissue showed a worsening in the overall score of the placebo group (P = 0.01), with emphasis on keratinized axis loss (P = 0.0001), hyperplasia (P = 0.001), homogeneity of the primary epidermal lamellae (P = 0.003) and hyperkeratosis (P = 0.05), while the adCTM group did not present a worsening in the overall score, with only an increase in mean stratum hyperplasia (P = 0.05). There was a difference between groups in the global post-treatment score (P ≤ 0.05), especially the absence of keratinized axis (P ≤ 0.01). The findings of this study demonstrated that the protocol of three intravenous injections of 2 x107 per regional perfusion in the digit of horses with stable chronic laminitis promotes improvements in vascular perfusion, hoof growth rate, spatial relation between hoof capsule / distal phalanx in the process scar tissue only on the hoof treated with adCTM, not verifying the same beneficial effect in the placebo group.