Three domestic short haired cats (2yrs, 5 yrs and 4 yrs of age) were presented with the history of large chronic, non healing wounds on the right lateral cervical (Fig1.A), left caudal forearm (Fig1.B) and right lateral thoracic region (Fig1.C), respectively to the Small Animal Clinic-Out Patient Unit of Madras Veterinary College Teaching Hospital. The etiology of the wounds was trauma in two cases and thermal burn in one case. The cats were presented two weeks after the injury and were treated by local vet before.
Physical examination revealed yellowish colored wound with putrid odour and purulent discharge. The open wounds were on the right lateral cervical, left caudal forearm and right lateral thoracic region respectively. The subcutaneous tissues were exposed; necrotic tissues and foreign debris were noticed. Moderate pain was evinced on palpation of the wound. The cats were in good health and the body temperature, mucous membrane color and capillary refilling time were normal. Blood samples were collected for hematology and serum biochemical analysis and also for preparation of Platelet Rich Plasma (PRP).
Treatment and Outcome
In all the three cases, mechanical debridement of the open wound was done by lavaging with sterile normal saline- 500 ml (Normal saline, 0.9 % NS, Baxter healthcare, USA) mixed with amikacin sulphate (250 mg, Alfakim, Ranbaxy, India) with a 20 ml syringe fixed with a sterile 22 gauge needle to produce a lavaging pressure of 22 psi.
Autologous platelet rich plasma was prepared by collecting 5 ml of whole blood via jugular venipuncture in K2 EDTA 5.4 mg vacutainer. The first centrifuge was performed at 100g at 40C for 10 min to separate RBCs from the remaining whole blood volume in a cold centrifuge. After the first centrifugation step, the whole blood separated into three layers: an upper layer that contained mostly platelets and WBC's, an intermediate layer that is known as the buffy coat and that was rich in WBCs, and a bottom layer that consisted mostly of RBCs. The upper half of the column which contained platelets, buffy coat and some RBC's was transferred to a falcon tube. The second centrifuge was then performed at 400g at 40C for 10 min. The upper portion of the volume that is composed mostly of PPP (Platelet-Poor Plasma) was removed to create the final volume of 1 ml of PRP (Platelet-Rich Plasma) (Fig 2). The PRP was activated by adding calcium gluconate 10% to the PRP in a 1:2 ratio and gently blended in a sterile Petridish until it reached a gelatinous consistency. The average yield of platelets were 770 × 10 3 /µL. It was then applied on the open wounds impregnated with Chlorhexidine gauze on days 0, 3, 7 in all the cases and bandaged. Wound planimetry was performed on days 0, 7, 14 by analyzing the captured photos into the "Image J" software which is used to measure the area of the open wounds. Based on this the percentage of epithelialization, wound contraction and healing process was calculated (Table 1). Histological evaluation of the open wound was done on days 7 and 14. After a healthy granulation was formed on the recipient wound bed, transposition flap reconstructive surgery was performed on third week of case presentation in all the cases.
Pre-operative antibiotics and analgesics were administered. The cats were premedicated with Diazepam-0.2 mg/kg (Lori, Neon laboratories, Mumbai, India) B.W intravenously and Butorphanol tartrate- 0.2 (Butodol-2, Neon laboratories, Mumbai, India) mg/kg B.W intramuscularly. General anaesthesia was induced with Propofol- 4 mg/kg (Neorof, Neon laboratories, Mumbai, India) B.W intravenously and maintained with Isoflurane- 2% (Forane, Abbott pharmaceuticals, India) in oxygen using Boyles anesthetic apparatus. Thesurgical areas were prepared aseptically. Flap planning was done meticulously (Fig 3.A and 3.B) . The area of the wound was measured in all the cases and flap size was calculated (Table 2). The base of the flap was equal to the width of the defect and the length of the flap was equal to the length from the outer pivot point to the most distant edge of the defect. The donor flaps of the respective cases were marked using a sterile felt tipped marker pen. The flap edges were incised and undermined in a stepwise fashion until the flap could be transposed into the defect with minimal tension. The flap was sutured into position by placing few walking sutures followed by intra dermal sutures with Polyglycolic acid 3-0. Skin was sutured with Polyamide-2-0 in simple interrupted suture pattern. A small corrugated drain tube was placed at the dependent part of the flap and sutured to provide adequate drainage of tissue fluids. The surgical wound was bandaged with an antibiotic ointment every 3 days. Post-operatively, Ceftriaxone- 10 mg/kg (C-tri, Intas Pharmaceuticals, Ahmedabad, India) B.W q12h, Tramadol- 2 mg/kg (Supridol, Neon laboratories, Mumbai, India) B.W q8h and Cetrizine- 0.1 mg/kg (Cetzine, Cipla, Mumbai, India) B.W q8h per-orally was administered for 7 days.
Complete flap uptake was observed in right lateral thoracic defect cat and the caudal forearm defect cat. (Fig 4.C and 4.B) The right cervical flap had partial uptake, except for a single 1 square cm region which was allowed to heal by second intention (Fig 4.A). Sutures were removed on 10th day post operatively. The cats had an uneventful recovery.
A color flow Doppler ultrasound imaging was performed using 7.5 MHz linear probe, on the donor area of skin flap before surgery and also post-operatively on days 3 and 7 to assess the blood flow pattern and perfusion (Fig 5).
Histopathological evaluation of samples taken from the corner of the wound on day 7, and 14 revealed early granulation tissue formation, angiogenesis and epithelialisation when compared to normal wound healing in the control animal, thus describing the efficacy of platelet rich plasma.
Skin wounds are commonly seen in veterinary practice. Open wound management, until the wound is considered suitable for reconstruction or until it has healed by second intention, has been the treatment of choice for centuries.9 Frequent and painful dressing changes over prolonged periods intensifies overall case management, and may impact treatment costs.2 Moreover, repeated sedation or short anaesthesia is often necessary for dressing changes, discouraging early ambulation and rapid recovery.
Platelet Rich Plasma (PRP) has the supreme advantage when compared to a single growth factor as it offers a multiple synergistically working growth factors that promoted mitogenesis of mesenchymal cells at the wound site.1 PRP is a concentrate of cytokines and growth factors (GFs) released from platelet alpha-granules at the site of tissue injury.6 Growth factors like transforming growth factor-B, platelet derived growth factor and vascular endothelial growth factor were released after platelet degranulation5 which in turn led to fibroblast recruitment, proliferation and matrix remodeling. Ultimately good granulation tissue formation and wound healing was noticed in all the above cases. The increased concentration of growth factors within the autologous PRP would be ideally suited for wound healing in dogs and cat.3 PRP application on open wounds in cats showed beneficial effects in wound healing by early promotion of granulation tissue, wound contraction and epithelialisation in all the above cases which was evident by wound planimetry histopathological evaluation.
In all the above cases, the number of platelets in the final solution was on average 4.5 times the baseline intravascular platelet count. That is, it was four to five times higher than the baseline count, which is considered the minimal concentration required to accelerate epithelialization and granulation.7 No systemic or local drugs were used after PRP application. Non-steroidal anti-inflammatory drugs (NSAIDS) were avoided, as they would have inhibitory effect on the treatment.
Transposition flap is a local pedicle flap that transposes skin from an area adjacent to the defect that is to be closed. It is a rotating subdermal plexus flap and it is supplied by terminal branches of the cutaneous arteries and veins.4 The width of the flap equals the width of the defect. The length of the flap is determined by the pivot point of the flap to the most distant point of the defect. The flap can be rotated into different angles.8 The most common angle of rotation is 90º and was followed in all the above cases. Trauma causes damage to the cutaneous vessels of the skin around the wound. Hence it is important to assess the integrity of the cutaneous vessels by Doppler ultrasound before planning an axial pattern or a subdermal plexus flap to reconstruct a traumatic wound.11
Color Doppler ultrasound technique was relatively easy, non-invasive, and inexpensive method of assessing the integrity of cutaneous arteries and veins.10 It also helped in assessing the blood flow pattern and perfusion in all the above clinical cases.
PRP is safe, fast, cheap, and simple to produce, and can be used to treat acute and sub-acute wounds in high motion areas like limbs of cats, as well as in cases of delayed wound healing and long-term non healing wounds. There are reports indicating the efficacy of platelet rich plasma in open wounds in dogs and horses. This is the first report of application and clinical use of PRP followed by reconstructive surgery in cats to promote healing in chronic wounds.