Effect of Intermittent Pneumatic Compression (IPC) on Healing of Immobilized Hemi-transected Achilles Tendonin Dogs A thesis Submitted to the Council of the College of Veterinary Medicine at the University of Sulaimaniin partial fulfillment of the requirements for the degree of Master of Sciencein Veterinary Surgery By DedanMohammadTaqiM.Amin BVM&S (2004)&DVS (2011), University of Sulaimani
Supervised By BahjatTaifor Abbas (PhD) Professor of Surgery
Xarmanan 2714
July2014
Acknowledgment Thanks to Great Allah, who gaves me a power and made all things possible Thank to my family for their warm, kind encourages, and moral support.
I would like to thank my supervisor, Professor Dr. Bahjat Taifor Abbas for his expert advice, brilliant comments, suggestions and encouragement throughout this study, who helped create the success that I continue to carry on today. He is not only served as my supervisor, but also encouraged and challenged me throughout my academic program, never accepting less than my best efforts. What is collected in this research are materials that I found them difficult but he makes me claim to obtain them. I would like to express my very great appreciation to the dean Dr. Umeid U. Uthman, academic staff, and the technical or support staff in the college of Veterinary Medicine and veterinary Teaching Hospital. I would like to express my sincerest gratitude to the Ministry of Agriculture and Water Resources, especially to the director of the Directorate of Animal Wealth & Veterinary, Dr. Abbas A. Abdi, for allowing me to complete my higher study and I extend my sincere thanks to the director of the Veterinary Directorate in Sulaimni, Dr. Aree S. Maeruf for his help and facilities for the period that followed the end of the study permission, that without it was impossible to complete my study. My gratitude also goes to the head of laboratory Dr. Kamaran M. Amin and his staff at the Directorate of Veterinary in Sulaimnai for their help and taking the time out of their busy schedule.
I should not forget my deepest thanks for the assistance given by my colleague Dr. Hazhar A. Hassan who shared with me for getting some of the street dogs used in this study at any time I asked him. Finally, I wish to thank various people for their contribution to this study; Mr. Muhammad, Mr. Soran and Mr. Jaza, for their valuable technical support.
DEDAN
Dedication
To my brothers, sisters and my sweet and loving mother, she prays of day and night makes me able to get such success and honor.
DEDAN
Summary
Achilles tendon (AT) injuries account as a common surgical affection in dogs and their healing is slow due to their low vascularity. Treatment of an injured AT is usually accompanied with an initial period of hind limb immobilization, which is detrimental to the healing process, partly by a reduction of blood circulation. Intermittent pneumatic compression (IPC) has been proposed to enhance tendon repair by stimulation of blood flow. The purpose of this study was to recognize the dynamic role of the IPC on healing of hemi-transectioned AT in dogs. It is hypothesized that daily IPC treatment can counteract the deficits caused by 21 days of immobilization post tendon hemi-transection. This study was carried on eighteen dogs. In all animals , hemi-transection of the right Achilles tendon was carried out. The study was conducted on the right AT , which were routinely prepared for asceptic surgery. Under the effect of general anesthesia, the right AT was exposed surgically and 1/3 to 1/2 of the full thickness of the tendon was transected and then reattached by suturing. The ends of the tendon was reattached with 3-0 nylon and sutured by Kissler suture pattern. The animals were then randomly devidid into two groups (control and treatment groups). In the control group, plaster of Paris was used to immobilize the limb with the further treatment. External fixator device was used for immobilization in the treatment group with daily application of IPC. The animals were authanized at 7, 14, 28 days after the operation. Gross and histopathological changes were studied. The necropsy result revealed for CG the hemi-transected AT thickened along their length and adhered to the surrounding subcutaneous tissues, while in the TG the heald tendons were grossly appeared much uniform in shape, none adhered ans less or without thickness due to the effect of the IPC treatment and the histological results revealed the progression of the healing stages in the repaired tendon in the TG by the effects of IPC while the stages of the tendons healing of the CG was progressed normally. According to the results of this study, intermittent pneumatic compression (IPC) treatment have an effect in accelerating the healing, and it can be used in conjunction with surgery to repair a cut or tear in the tendon. ii
Contents Summary ....…………………………………………………….……….………….… ii Contents ........................................................................................................................ iii List of figures ……………………………………………………………………….… v List of abbreviations …………………………………….…………………..…..……. viii
Chapter one Introduction ………………………………..……………………….…….………… 1 Chapter Two: Review of Literatures 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. 2.8. 2.9. 2.10. 2.11. 2.12.
Anatomy of Achilles tendon …………………………………………..…..…… 3 Histology of Achilles tendon................................................................................ 6 Causes of tendon injury ........................................................................................ 7 Clinical signs of rupture of the Achilles tendon ……………………………….. 7 Tendon healing ………………………………………………………….…….… 8 Pathophysiology………………............................................................................ 9 Diagnostic imaging ……………......................................................................... 10 Tendon surgery: principles and techniques …………………………….…..… 10 Fixation of the limb and recheck examination ...………………………….… 12 Postoperative management ................................................................................. 14 Biophysical characteristics of IPC ………………………………………….… 14 Mechanismof action of intermittent pneumatic compression (IPC) ................... 15
Chapter Three: Materials and Methods 3.1 3.2.
3.3. 3.4. 3.5.
Materials ………………………..……………………………………………… 17 Methods ……………………………………………………………..…………. 18 3.2.1. Animal group …..................................................................................... 18 3.2.2 Plan of the work……………………………………………………. . 19 3.2.3. Surgical Procedure for Achilles tendon hemi-transection......... ............. 20 3.2.4. Immobilization techniques for CG and TG ………………………...…. 22 Intermittent pneumatic compression (IPC) treatment ….………………..…….. 22 IPC procedure ………………………………………………………......……… 24 Clinical follow up for AT hemi-transection …………………….…...….……... 24 iii
3.6.
Histopathology examination ……………………………………….…….…… 25
Chapter Four: Result 4.1.
Anesthetic effects ……………………………………………………..…..………………………………… 26
4.2.
Postoperative fixation of hock Joint ………………………..……………...… 26
4.3. 4.4.
Post operative clinical signs ………………………..……...………….….… 28 Gross Achilles tendon findings ………………………………………….….. 29
4.5.
Pathology for Achilles tendon healing ………………………………..…..… 33 4.5.1. The 7th P.O. days ………………………………………….………... 33 4.5.2. The 14th P.O. days ………………………………...………..……….. 35 4.5.3. The 28th P.O. days …………………….…..….………………….…... 37
Chapter Five: Discussion 5.1. 5.2. 5.3. 5.4. 5.5.
Anesthesia …………………………………………………………………… 39 Achilles tendon tenorrhaphy ……………………………………………….. 39 Postoperative fixation of hock Joint …………………………………………………………….. 41 Effect of IPC treatment………………………………………………………………………………… 43 Histological finding of repaired AT………………………………………………………………… 45
Conclusion and future study ………….……………………………..……………..… 46 References …………………………………………………..………..……....…...…… 47
iv
List of Figures Figure No.
Figure Title
Page No.
Figure 1
Posterior view for canine pelvic limb masculature .........................
5
Figure 2
The gastrocnemus muscles ………………………………………..
5
Figure 3
Schematic view of modified Kissler (Piskin et al., 2007) .............. .
13
Figure 4
clipped and shaved from the mid-shaft of the tibia ………………
20
Figure 5
Achilles tendons were dissected free from the surrounding tissue ……
Figure 6
The Achilles tendon hemi-transection which involved 1/3 -to- 1/2 of its full thickness…………..……………………
Figure 7
Figure 8b
The Unilateral External Fixator Device …….………
21 23
Schanz type self-drilling pins were fixed above and below the hemi-transected tendon tendon ………………………….
Figure 9
21
The hemi-stranected Achilles tendon was sutured by locking-loop suture pattern, using 3-0 nylon suture material …
Figure 8a
20
23
IPC is applied directly above the repaired Achilles tendon in one of the dogs of the treatment group dogs ………
25
Figure 10
IPC device shows 12 mmHg pressure applied to the repaired AT ……
25
Figure 11
The image A shows proper healing of the Achilles tendon on the
Figure 12
28th day P.O. ………
28
Post-Mortem for a dog from control group on the 7th P.O. day. .…
29
v
Figure 13
Post Mortum for a dog in the treatment group on the 7th P.O. days …
Figure 14
The image of gross Achilles tendon distortion that was causing partial restriction in limb movement ……
Figure 15
30
A photograph of gross Achilles tendon for a dog (No. 9) in the treatment group on the 14th, P.O. day. ……….……………
Figure 16
30
The gross Achilles tendon in a dog No. 7 from the control group on the 28th P.O. day ……………………
Figure 17
31
Post-Mortum for a dog (No. 2) from the control group on the 28th, P.O. ………………
Figure 18
29
31
Image of the dog No.1 of the treatment group on the 28th P.O. day showing the repaired Achilles tendon uniform and cord-like in shape. ..
Figure 19
32
Post-Mortum, of the repaired Achilles tendon (dogs No. 1) from the TG on the 28th day P.O. was healed properly and appeared cord-like in shape without adhesions toits surrounding tissues….
Figure 20
A micrograph of dog´s Achilles tendon of the control group on the 7th P.O. day. ……..
Figure 21
34
A micrograph of repairing Achilles tendon from the treatment g on the 7th day. ……
Figure 22
32
34
A micrograph of repairing Achilles tendon on the 14th day in the control group showing. ………
36
Figure 23 A micrograph showing repairing Achilles tendon on the14th, day in the treatmnent group. ………… vi
36
Figure 24
A micrographof repairing Achilles tendon on 28th days in the control group. ………………..…
38
Figure 25 A micrograph of repairing Achilles tendon from the treatment group on the 28th P.O. day. ……
vii
38
List of abbreviations
ArtAssist® AT CCT CG cm DVT ECM EFD G GT GTO H&E IM IPC Kg Ml mmHg NO P.M. P.O. PE POP SDF SQ TESF TG USP
Arterial Art Assist Achilles tendon Common calceneal tendon Control group Centimeters Deep vein thrombosis Extracellular matrix External fixator device Gauge Gastrocnemous tendon Golgi tendon organ Hematoxyline-Eosin Intramauscular Intermittent pneumatic compression Kilogram Millimeter Millimeter of mercury Nitric oxide Post mortem Postoperativilly pulmonary embolism Plaster of Paris Superficial digital flexor tendon Subcutaneous Transarticular external skeletal fixator Treatment group United States Pharmacopeia
viii
Chapter One
Introduction
Throughout the decades, a number of possible therapies have been experimented for treatment of injured tendons, yet none of them has ever turned out to be able to ensure a complete restoration of the anatomical and functional integrity of the injured tissue. Nowadays, scientific research has brought to light the positive effects on tendon healing by a process highly dependent upon the sense of adequate blood flow stimulation. Injuries to tendons are a major problem for any kind of animal and often demanding high efforts to improve the quality of the healing process of these tissues and the aim usually was to obtain a scar tissue enough strong and elastic to allow somehow the reintroduction to activity. Achilles tendon (AT) is the thickest and strongest tendon in the animal body. Like any other tendon in the body, however, it is susceptible to over use and rupture (Gebauer, 2007). One of the main requirements for the reconstruction of the tendon is to achieve adequate tensile strength. Modern tendon repair aims to achieve gliding and to restore function as soon as possible. More recently, some surgeons have advocated for a more rapid progression and return to activity.This has led to the introduction of several treatment modalities to enhance healing and expedite recovery. Intermittent pneumatic compression (IPC) is one of them. It is a recent treatment method used in several human and animal studies hypothesized to decrease venous stasis and stimulate blood flow that exert positive effects on tissue healing, a process highly dependent upon adequate circulation. IPC is used in the management of fractures, tendon and softtissue injuries, but its potential role in tissue healing has also been investigated (Challis et al., 2007; Kannus et al., 2000; Park and Sliva, 2003). The mechanical effect of IPC is the improvement in local vascularity and its daily uses enhance neurovascular ingrowths and fibroblast proliferation in the healing tendon and may accelerate the repair process (Dahl et al., 2007).
1
In a randomized trial comparing routine wound care plus compression versus routine wound care, compression and IPC, the results clearly demonstrated that the addition of IPC improved over all healing and healing rates (Nikolovska et al., 2005). Tendons adapt to changes in mechanical loading, and numerous animal studies show that immobilization of a healing tendon is detrimental to the healing process (Eliasson et al., 2012). For many years, patients were rigidly immobilized for 6–8 weeks postoperatively. New studies have shown excellent results with early weight bearing, and this is quickly becoming the standard of care amongst many physicians (Khan et al., 2005). The goal of accelerated rehabilitation is to prevent muscle atrophy and joint stiffness that worsen the longer the joint is immobilized.
Aim of study The study was done to evaluate the effect of daily treatment of IPC for 4-28 days on healing of hemi-transected Achilles tendon in dogs.
2
Chapter Two Review of Literatures 2.1. Anatomy of Achilles tendon (AT) Healthy tendons are brilliant white in color and have a fibroelastic texture. Tendons demonstrate marked variation in form; they can be rounded cords, strap like bands, or flattened ribbons (Michell and Watkins, 1993). Tendons are varying in length and thickness, sometimes round, sometimes flattened, and devoid of elasticity. They consist almost entirely of white fibrous tissue, the fibrils of which have an undulating course parallel with each other and are firmly united together so that they transmit the force produced by muscle to bone and also prevent muscle damage by acting as shock absorbers (Levangie and Norkin, 2001). Due to the thickness of its fibrous tissue, a tendon provides joint stability at the articulation; and crossing joint surfaces is often encased in a tendon sheath to facilitate movement during joint motion (Fossum et al., 2004). Two types of tendon are a vascular tendon, which includes those tendons with a tendon sheath (e.g. the digital flexors). These tendons are considered to have a poor blood supply and result in poor healing ability, with increased risk of adhesions between the tendon and the sheath. However, the vascular tendons have a much better defined, vascular supply.The blood vessels enter the tendon only at specific points along the tendon. Vascular tendons include those tendons without a tendon sheath that are surrounded by muscle and soft tissue, for example the Achilles tendon or the triceps tendon. In unsheathed tendons, vessels may pass through the surrounding paratenon into the tendon at any point along the tendon. The tendon innervations originate from cutaneous, muscular, and peritendinous nerve trunks. At the myotendinous junction, nerve fibers cross and enter the endotenon septa. Tendon is a comparatively poorly vascularised tissue that relies heavily upon synovial fluid diffusion to provide nutrition (Fenwick et al., 2002). The blood supply to the tendon is from three areas: the musculotendinous and osseotendinous junctions and the paratenon, with the posterior tibia artery providing the major contribution (Gillbert et al., 2007). Nerve fibers form rich plexuses in the paratenon and branches penetrate the epitenon. Most nerve fibers do not actually enter the main body of the 3
tendon, but terminate as nerve endings on its surface. The nerve endings of myelinated fibers function as specialized mechanoreceptors to detect changes in pressure or tension. The Golgi tendon organ (GTO) is located at the musculotendinous junction. During muscle contraction, this junction stretched (Lamb and Duvernois, 2005). These mechanoreceptors, the Golgi tendon organs, are most numerous at the insertion of tendons into the muscle (Lephart et al., 1997). Golgi tendon organs are essentially a thin, delicate capsule of connective tissue that encloses a group of branches of large myelinated nerve fibers. These fibers terminate with a spray of fiber endings between bundles of collagen fibers of the tendon. Unmyelinated nerve endings act as nociceptors, and they sense and transmit pain. Both sympathetic and parasympathetic fibers are present in a tendon (Ackermann et al., 2001). The Achilles tendon (AT) is the single largest, thickest and strongest tendon in the body that transmits the force of powerful gastrocnemius muscles in the foot, facilitating walking and running. The canine Achilles tendon, also known as the common calcaneal tendon (CCT), is composed of three distinct musculotendinous components which converge and insert on the proximal aspect of the calcaneus. The three structures are the gastrocnemius tendon (GT), the superficial digital flexor tendon (SDF), and the combined tendon (CT) of the gracilis, biceps femoris, and the semitendinosis muscle. The major component of this group (Figure 1) is the GT muscles which are paired, but unite in the upper part to form a single tendon, which inserts on the proximal dorsal surface of the calcaneus and is responsible for extending the tarsus. The SDF inserts collaterally at the proximal calcaneus before continuing distally and attaching to the digits in the second row of the phalanges forming a broad cap over the point of the hock (Figure 2). The last component of the CT inserts at the medial aspect of the calcaneus and also plays a minor role in the tarsal extension (Dyce et al., 2002; Daniel, 2006; Ying et al., 2003; Maitre et al., 2009). The AT is supplied by two arteries, the posterior tibial and peroneal arteries. Three vascular territories were identified, with the midsection supplied by the peroneal artery, and the proximal and distal sections are supplied by the posterior tibial artery (Chen et al., 2009) as determined by the small number of blood vessels per cross-sectional area, which do not in general vary significantly along its 4
length.Vessels at the bone insertion do not pass directly through from the bone into the tendon due to a cartilage layer between the tendon and bone, but they anastomose with those of the periosteum, forming an indirect link with the osseous circulation (Fenwick et al., 2002). The caudal saphenous artery is a fine branch entering the midbody of the tendon along its cranial border in canine CCT. The musculotendinous junction had additional branches from the gastrocnemius muscles. Distally, a traumatic handling and minimal manipulation should be used during the surgical approach and debridement to preserve the remaining blood supply in ruptured tendons (Gilbert, 2007).
Figure 1: Posterior view for canine pelvic limb masculature (Sasha and Ashely, 2009)
Gastrocnemeus muscle Saphenous Vein Achilles tendon
Calceneal tubercle
Figure2: The gastrocnemius muscles are paired and unite in the upper part to form a single tendon, which makes up the majority of the common calcaneal tendon. 5
2.2. Histology of the tendon The smallest structural unit in tendons is the fibril, which largely consists of strictly organized collagen molecules. Tendon fibrils make up the fibers. Fibers, in turn, are bundled to represent the fascicles, which are enclosed in the endotenon which supplies blood vessels and nerves to the tendon. Bundles of fascicles are surrounded by the epitenon, which is continuous with the endotendon. The collagen fibers are packed closely together, parallel to the direction of force. Vessels are generally arranged longitudinally within the tendon, passing around the collagen fiber bundles in the endotenon. The AT is surrounded by the paratenon, composed of types I and III collagen fibrils, elastic fibrils and a layer of synovial cells. Parateanon is thinner than the synovial sheaths surrounding other tendons but some tendons are enclosed by a synovial sheath with an outer fibrous layer, while the two inner layers of synovial sheaths secrete peritendinous fluid and act to decrease friction. (Jozsa and Kannus ,1997; Astrom and Westin, 1994; Fenwick et al., 2002; Sharma and Maffulli, 2005; Pasquini et al., 1997). The mesotendon (the middle layer of the paratenon) supplies most of the blood of the tendon, but is lower near the calcaneal insertion. During a contraction, blood flow to the tendon greatly diminished and can cease completely. The basic constituents of tendon fibers are collagens; proteoglycans, water, tynocytes and glycoproteins. Collagens provide the raw mechanical strength of the tendon. Collagen I is the dominating collagen subtype representing approximately 95 % of the total collagen, while collagen III is the second most common, representing ~3 % and collagen V is ~2 %. In normal tendons, type III collagen is mainly located in the endotenon and epitenon. However, it is also found in aging tendons
Proteoglycans,
e.g.
decorin,
biglycan
and
aggrecan,
carrying
glucosaminoglycans as side chains, link together and demarcate the collagen fibrils. They also provide water-binding characteristics and resist compressive load. This glycoprotein is found throughout the tendon and is thought to play a role in the organization and orientation of the extracellular matrix (ECM) within the extracellular matrix network, tenoblasts and tenocytes constitute about 90% to 95% of the cellular elements of tendons. Tenoblasts are immature tendon cells. They are spindle-shaped and have numerous cytoplasmic organelles, reflecting their high metabolic activity (Kannus et al., 2000). As they mature, tenoblasts become elongated and transform 6
into tenocytes. They appear as star shaped cells in cross sections and appear as rows parallel with the tendon fibers in longitudinal sections. It appears to be important for the elasticity and responds to mechanical load by increased synthesis, which is also regulated by growth factors and cytokines (Jarvinen, 2000).
2.3. Causes of tendon injury There are multiple mechanisms for tendon injury. Tendons typically fail by either acute mechanical overload, or by accumulated damage from repeated subclinical injury, repetitive damage includes exercise induced hyperthermia, hypoxic injury reperfusion injury and mechanical strain/stretching. Mechanical strain can also lead to a repetitive breakdown of the tendons. When strain is initially applied to the tendons, individual collagen fibers undergo elastic deformation and straighten out, losing their wavy appearance. When these strains are relieved, they resume their normal unstressed shape. Individual fibers demonstrate a linear, elastic response to 25% elongation, and then resume a normal wavy structure once the strain is removed, with resumption of normal length. When tendons are loaded as a whole unit, they demonstrate a linear response to a much larger amount of strain, as high as 20-50%. This is thought to be due to the three-dimensional organization of fiber bundles within the whole tissue (Jozsa and Kannus, 1997; Rochat, 2005; Fenwick et al., 2002). Injection directly into tendons results in necrosis due to pressure increase and hypoxia within the tendon, and also has substantial effects on tensile strength, modulus of elasticity and healing (Rewartt et al., 1997). The etiology of AT injuries in dogs is usually traumatic and depending on the trauma, the severity of the lesion may vary considerably leading to stretching (hyperflexion because of overstraining or extensive ankle flexion). Small or partial lacerations or a complete rupture usually occur in high jumps, traffic accidents or fighting (Clark, 2001; Montgomery and Fitch, 2003).
2.4. Clinical signs of rupture of the Achilles tendon Damage to a tendon caused by an external force is also termed strains. The degree of tendon involvement can vary from partial to complete. First-degree strains 7
or mild strains result from short-lived application of moderate force, with relatively few damaged fibers, and minimal functional effects. Second-degree or moderate strains are characterized by increased numbers of damaged collagen fibers and marked functional deficit, although the tendon is grossly intact. Third-degree or severe strains have actual interstitial disruption (partial or complete), with complete loss of function (Piermattei et al., 2006). The clinical signs associated with the rupture of the Achilles tendon will vary depending on the degree of injury, but the “Plantigrade position” and the swelling around the tendon insertion on the hock are the main symptoms observed in a dog with complete rupture of the tendon (Cervil et al., 2010).
2. 5. Tendon healing Tendon healing can occur intrinsically, via proliferation of epitenon and endotenon tenocytes, or extrinsically, by an invasion of cells from the surrounding sheath and synovium (Sharma and Maffulli, 2005). The intrinsic model produces obliteration of the tendon and its tendon sheath. Healing of the defect involves an exudative and a formative phase, which on the whole, are very similar to those associated with wound healing (Tillman and Chasan, 1996). Extrinsic healing occurs through the chemotaxis of the specialized fibroblasts into the defect from the ends of the tendon sheath (Wang et al., 2002). The process can be divided into three phases: inflammation, repair or proliferation, and remodeling. The initial, inflammatory phase lasts from 4-7 days in controlled injuries a prolonged inflammatory phase may negatively affect tendon healing and repair (Clark, 2001). Erythrocytes and inflammatory cells, particularly neutrophils, enter the site of injury. In the first twenty-four hours, monocytes and macrophages predominate and phagocytosis of necrotic materials occurs (Sharma and Maffulli, 2005). Vasoactive and chemotactic factors are released with increased vascular permeability, initiation of angiogenesis, stimulation of tenocyte proliferation, and recruitment of more inflammatory cells (Murphy et al., 1994). Tenocyte gradually migrate to the wound, and type-III collagen synthesis is initiated. After a few days, the proliferative phase begins with attraction of undifferentiated mesenchymal cells and production of the matrix and unorganized collagen fibers 8
(Fenwick et al., 2002). This phase begins during the inflammatory phase, and continues until approximately the 21st day. Initially, the collagen is mostly immature type III, with small diameter fibrils arranged in a haphazard fashion. By the 12-14th days, the type III collagen will begin to be replaced by type I collagen. Synthesis of type-III collagen peaks during this stage and lasts for a few weeks. Water content and glycosaminoglycan concentrations remain high during this stage (Oakes, 2003). In the repair phase, vascular ingrowths are needed to provide oxygen and nutrients to active fibrocytes. After approximately six weeks, the remodeling phase commences, with decreased cellularity and decreased collagen and glycosaminoglycan synthesis. This phase requires a slow but continuous loading of the tendon over time (Clark, 2001). The consolidation stage begins at about six weeks and continues for up to ten weeks. During this period, the repair tissue changes from cellular to fibrous. Tenocyte metabolism remains high during this period, and tenocytes and collagen fibers become aligned in the direction of stress (Oakes, 2003). Type I collagen begins to predominate again, with increases in fibril diameters and formation of stable crosslinks. Reorganization of collagen fibers parallel to lines of stress is first seen by 5-6 months after the initial injury. Decreases are also seen in the number of macrophages, fibroblasts, myofibroblasts, and capillaries, as well as decreased amounts of glycosaminoglycans. The collagen becomes much more densely packed, and becomes almost exclusively type I. This reorganization can take up to a year, and is associated with increasing strength (Clark, 2001).
2.6. Pathophysilogy The Achilles tendon does not have good blood supply or cell activity, so injuries can be slow to heal. The tendon receives nutrients from the tendon sheath or paratendon. When an injury occurs to the tendon, cells from surrounding structures Migrate into the tendon to assist in the repair. Some of these cells come from blood vessels that enter the tendon to provide direct blood flow to increase healing. With the blood vessels come nerve fibers. These nerve fibers are the cause of the pain (Alfredson et al., 2003; Jarvinen et al., 2000).
9
2.7. Diagnostic imaging: X-rays are useful to rule out the fractures, but are of limited value to diagnose Achilles tendon ruptures (Schaweitzer and Karasick, 2000). Although MRI shows the ruptured tendon, it is usually not necessary to obtain an MRI because the diagnosis is obvious to examination (Berkson, 2012). The Achilles tendon is examined above the calcaneus for swelling and continuity (Swiderski et al., 2005). Ultrasonography can also be used as a diagnostic tool. It is especially useful to differentiate partial and complete ruptures, as well as for monitoring during the course of tendon healing. Normal tendons have a very characteristic architecture, with the mid-portion of the tendon appearing as an echogenic structure with parallel, hyperechoic lines, surrounded by a hyperechoic, thick, smooth band (peritenon). Abnormal findings in partial tendon ruptures include thickening of the calcaneal tendon, with the loss of the normal linear pattern. The echogenicity is typically inhomogenous. With complete rupture, complete interruptions in the tendon structure can be noted, with the loss of the normal tendonous echo-structure. Hematoma formation can be evident, with an anechoic, inhomogenous, irregularly delineated area present within the gap. During the healing process, inhomogeneity decreases, and the typical fibrillar structure of the tendon reappears (Rivers, 1997). Ultrasound therapy may be able to accelerate healing and tendon maturation in dogs as well (Saini et al., 2002).
2.8. Tendon Surgery: principles and techniques The principles of tendon repair are based on traditional practices, clinical experience and interpretation of experimental animal studies. An understanding of the anatomic features of the tendon, the nutrient pathways to the tendon, and the physiology of the healing process is essential to the formulation of a conceptual approach to tendon repair (Paule, 2001). Obviously, absolute asepsis is essential. However, other factors which may determine infection such as dead spaces, buried foreign bodies (including excessive suture material and ligature), closure with insufficient hemostasis, excessive separation of tissue layers, drying of tissues, and long surgical exposure of tissues (Jenson et al., 2005; Fossum et al., 2004). 10
Of equal importance in Iimiting complications is the reduction of tissue trauma. Tissue forceps should hold more by retracting than by grasping to prevent crushing or torn tissue. More specifically, the tendon, its sheath or paratenon, and its gliding mechanism must be preserved (Jenson et al., 2005). At no time should the tendon be grasped with forceps or wrapped in gauze. The only place that a tendon should be handled with forceps is in an area that will either be removed or where the creation of an adhesion is desirable. At no time should the tendon or the surrounding tissues be allowed to dry. Therefore, saline and suction should be used to adequately visualize the field if minor bleeding occurs. Incision planning must be carefully considered to reduce wound trauma and prevent fibrosis, longer area of the tendon; however, this tends to create a longer area of scar tissue and an increased possibility of long binding scars that inhibit function. Therefore, an incision should be transverse to the longitudinal axis of the tendon if it is feasible to perform the surgery with that limited exposure. If not, the primary incision should be made parallel to the tendon at some distance through the skin and then perpendicular through any underlying soft tissues. At no time it was thought that incisions should be longitudinal to provide better exposure over damage to the surrounding tissues that make up the so-called gliding mechanism. Tendon anastomoses should be placed in a sufficient tissue bed. Thus, subcutaneous tissue must be preserved to protect gliding function (Fossum et al., 2004). There are two options for treatment; one option is an operation to repair the tendon surgically and the second is conservative. Surgical treatment is preferred over conservative medical management in cases of complete gastrocnemius tendon rupture (King and Jerram, 2003; Shani and Shahar, 2000).The proper choice of suture material is particularly important. The mechanical properties of the suture material resemble the properties of the reconstructed tissue (Jenson et al., 2005). The ideal tendon suture should have high tensile strength, should tie in such a way that extra knots are not required, nonabsorbable monofilament suture in order to allow gliding motion along the suture, and should not create a reaction within the tissue but sustain reapposition of the tendon ends for at least three weeks (King and Jerram, 2003). Nylon or polypropylene is preferable; however, steel wires, polidioxanone or polyglyconate may be also used. The recommended suture size is 3-0 (USP scale). The use of larger suture material could negatively affect the healing process by increasing tissue reaction. Also, the suture should not obstruct the local blood supply 11
and provoke diastasis of the tendon ends (Fossum et al., 2004). The anatomical arrangement of parallel fibers in tendons with few cross-linking fiber provide little to prevent suture material from slipping and loosening towards the tenotomy when load is applied. There are specific suture patterns that are designed to hold in tendons and prevent gap formation under load: the 3-loop pulley, Kessler locking loop, and Krackow suture patterns being three examples (Montgomery and Fitch, 2003). The Kessler locking loop (Figure 3) and Krackow sutures are considered for use more in flat tendons. The 3-loop pulley is considered better used in round tendons. Moores et al., (2004) compared the 3-loop pulley to 2 locking loop sutures, and found that the 3loop pulley was more resistant to gap formation, although both had similar forces at ultimate failure. The 3-loop pulley has also been found to be almost twice as strong as a single locking loop suture in preventing both 1 mm and 3mm gap formation in Achilles tendon repairs under tension, and the choice of pattern is based on the anatomical configuration of the tendon.
2.9. Fixation of the limb and recheck examination During the preoperative period, tendon immobilization is necessary to prevent end separation (Fossum et al., 2004). Also primarily in the early stages of healing, limb immobilization is essential because movement can produce a significant space between the tendon ends, decreasing the local blood supply and increasing fibrosis, which can compromise healing and final functional outcome (Clark, 2001) allows restoration of about 50% of normal tendon strength, but results in significant muscle weakness.
12
Figure 3: Schematic view of Modified Kessler (Piskin et al., 2007).
Immobilization techniques include transarticular external skeletal fixation (TESF), placement of a calcaneo-tibial bone screw, and various configurations of splints and casts (Guerin et al., 1998; Nielsen and Pluhar, 2006). Immobilization should not be for more than six weeks. In fact, joint fixation for more than three weeks could cause varus or valgus deforrmities (Fossum et al., 2004). suggests complete postoperatively (P.O.) immobilization for three weeks, followed by a three-week partial immobilization, allowing an acceptable restoration of function and reducing the risk of a new lesion. The first recheck should occur at 7-10 days. In addition, a patient with an external fixator should be rechecked at 6-8 weeks, then every 3-4 weeks thereafter. If a full-length limb bandage is to be placed, the middle two digits should be left exposed, in order to monitor for discomfort, or any sign of reduced circulation (Fossum et al., 2004). At this time sutures, if any, should be removed. Some distal limb edema should be expected, but this should have decreased over the last few days and not be getting worse. The patient should be rechecked if stops weight bearing, or if there is excessive bleeding or excessive discharge at the pin sites. The recheck examination should include a brief general physical examination, including temperature, pulse, and respiratory rate. All pin sites should be evaluated for pain by gentle palpation. Some serosanguinous discharge is to be expected. Purulent discharge is abnormal. 13
The amount of discharge should not be so great that the bandage becomes so saturated that it needs to be changed more often than twice per week or that discharge drips down on none bandaged limb (Kraus et al., 2003). Fossum et al., (2004) suggests that after 3–6 weeks of fixation, the limb should be supported with a padded bandage to prevent dorsal tarsus flexion, and activity must be restricted to walking on a leash for 10 weeks.
2. 10. Postoperative management Protection of the repair early in the healing process is a must. Additional support can be from a cast, external skeletal fixator, splint, or calcaneal-tibial bone screw. All of these methods provide relief of tension on the repair of the tendon for three weeks to three months after surgery (King and Jerram, 2003; Nielsen and Pluhar, 2006). Many practices support the repaired tendon with an orthotic such as a neoprene brace, hinged splint, or other custom-made device so that the dog may again begin training as soon as possible. Immobilization for longer than four weeks will result in deleterious effects on the joints, some of which can be permanent. In addition, early mobilization of the joint improves the healing process and augments the tensile strength of the tendon repair (Virchenko and Aspenberg, 2006; Enwemeka, 1992; Murrell et al., 2009). A dog may achieve a stable, functional hind limb after repair, but return to competition is less likely than return to function as a pet. A study from New Zealand determined that only seven of 10 dogs returned to full or substantial levels of work after healing, and 29% of those had moderate persistent lameness (Worth et al., 2004).
2.11. Biophysical characteristics of IPC Intermittent pneumatic compression (IPC) is a therapeutic technique used in medical devices that include an air pump and inflatable auxiliary sleeves, gloves or boots in a system designed to improve venous circulation in the limbs of patients who suffer edema or the risk of deep vein thrombosis (DVT) or pulmonary embolism (PE). 14
In use, an inflatable jacket (sleeve, glove or boot) encloses the limb requiring treatment, and pressure lines are connected between the jacket and the air pump. When activated, the pump fills the air chambers of the jacket in order to pressurize the tissues in the limb, thereby forcing fluids, such as blood and lymph, out of the pressurized area. A short time later, the pressure is reduced, allowing increased blood flow back into the limb. The primary functional aim of the device “is to squeeze blood from the underlying deep veins, which, assuming that the valves are competent, will be displaced proximally.” When the inflatable sleeves deflate, the veins will replenish with blood. The intermittent compressions of the sleeves will ensure the movement of venous blood (Turpie, 2007; Urbankova, 2005).
2.12 Mechanism of action of intermittent pneumatic compression (IPC) The type of IPC among several types and its mechanisms of action are: First, compresses the foot and ankle. One second later, the calf is compressed. As a result, the foot, ankle and calf veins are almost completely emptied. In return, the arterial blood is more easily pushed down to the toes and blood-deprived tissues. Because of this mechanism, blood flow to the skin of the feet can be tripled (Nicolaides, 1997). A second mechanism of action that accounts for the large blood flow increase involves the endothelium (cells that comprise the lining of all blood vessels). Endothelial cells release important biochemical factors, such as nitric oxide (NO) that helps circulate the blood (Kearon et al., 2008). After approximately 12 seconds, the device repeats the compression sequence one time per minute this also appears to support the other hematologic aspects observed with intermittent pneumatic compression (Eze et al., 1996).
15
Chapter Three
Materials and Methods
3.1. Materials
1. Animals: Eighteen local breed street dogs from both sexes were used. They were captured randomly from different localities of Sulaimani province. Their body weight and age ranged between 7.4 to 14.0 kg (12.2 ± 4.9 kg), and around 7-13 months (9.4 ± 2.0 months) respectively, and were hosted in individual cages at the Animal House under the same veterinary supervision, in the Veterinary Teaching Hospital - College of Veterinary Medicine/ University of Sulaimani. The dogs were physically healthy and free from orthopedic and neurological diseases, congenital and acquired abnormalities. They were allowed at least for oneweek under undisturbed acclimatization before initiation of the experiments. Dogs were maintained with free food and water access. 2. Surgical instruments: included instruments for orthopedic surgical set. 3.
External
Fixation
Device
(Synthes®
Digital
multinational medical device). 4. Suture materials; Nylon 0/3 and Vicryl 0/2. 5. Drugs: a. Ketamine HCl 10% (Alfasan, Woerden-holland) b. XylazineHCl 2% (Alfasan, Woerden-holland) c. Atropine Sulphate 1mg/ml (Cenavisa Labratories- Spain) 16
Radius
Fixator
set;
d. Dexamethazone0.2% (Colvasone, Norbrook Laboratories (GB) Limited) e. L-SPEC 5/10 (Lincomycin 50 mg - Spectinomycin 100 mg/ 1 ml; D.V.M. Belgium. f. Dipyron 500 mg/ml (Vapco - Jordan) g. Vitamin - B - Complex (Interchemie - Holand) h. Ivermectine 1% (Alfasan, Woerden-holland) i. Oxytetracycline Spray (OTC, Daru darman Co., Tehran. 6. Intermittent pneumatic compression (IPC) device (Medical Rossmax Blood Pressure Monitor); specifications: pressure: 40
250 mm Hg; pulse: 40 - 199
Beats/minute; inflation pump driven; deflation: Automatic Air Release Valve; Accuracy: pressure: ± 3mmHg; pulse: ± 5% of reading. 7. Study approval This study was approved by the Vet College Committee for Animal Ethics and Care at College of Veterinary Medicine / Sulaimani University and carried out at The Department of Surgery and Theriogenology.
3.2. Methods:
3.2.1. Animal groups Following repair of the AT hemi-transection in the eighteen dogs, the animals were randomly divided into two equal groups CG and TG (9 dogs per each). Additionally, both CG and TG groups were randomly and equally alienated into 3 subgroups: I, II, and III (i.e., 3 dogs for each).
17
3.2.2 Plane of work
18 Dogs
9 Dogs TG
9 Dogs CG
AT hemi-transection
Immobilization by POP
Immobilization by EFD
No treatment
IPC treatment
Euthanization after:
Euthanization after
14days
7days
28days
7days
28days
14day s
O
O
O
O
P
P
P
P
O O P
Post-mortem and histological examination
18
P
3.2.3. Surgical Procedure for Achilles tendon hemi-transection Random assignment of dogs for surgery to the right pelvic limb was determined prior to the start of the study. Premedication included subcutaneous (SQ) injection of atropine sulfate (0.02- 0.04 mg/kg). Induction of anesthesia followed after 30 minutes by intramuscular injection of xylazine (2 mg/kg) and ketamine hydrochloride (10 mg/kg) mixture. The animals were kept anesthetized with intermittent intramuscular applications of ketamine and xylazine in half doses (Mckelvey and Hollingshead, 2003). Hair was removed (clipped and shaved) from the lateral and medial aspects from mid-shaft tibia down to the proximal phalanx of the right pelvic limb (Figure 4). The operation field for dogs were surgically prepped with chlorhexidine scrub followed by a wash with water, and finally was swabbed with povidone iodine 10%. The AT was exposed through a posterior 10 cm skin incision, and lower part of Achilles tendon was dissected free from the surrounding tissue. Appropriate tension on the tendon was maintained to achieve a neutral position and to stabilize the tendon. During suturing, two sterile needles (22G × 1¼") were passed transversely through the proximal and the distal tendon ends were inserted into AT stump, about 5 cm apart from each (Figure 5). The site of transaction was 5-7 cm proximal from the right tendon’s vertex of calcaneus. The hemi-transection involved 1/3 -to- 1/2 of the full thickness of the AT (Figure 6). Achilles tendons were sutured by locking-loop suture pattern (Tomlinson and Moore, 1982), using nylon suture material size (USP 3-0). Each singular component of the severed AT was identified and individually involved within the suture (Spinella et al., 2010). Marginal points of tissue entry for transverse parts of the suture were placed 2.5 cm away from the hemi-transection (Figure7). Systemic antimicrobial therapy (L-Spec 5/10 at the dose rate of 2ml/kg), Vitamin –B- complex (2ml/kg) and dipyron (2ml/kg) were given for 3 days intramuscularly (IM).
19
Figure 4: The hair was removed (clipped and shaved) from the mid-shaft of the tibia to the proximal phalanx of the right pelvic limb.
Figure 5: Achilles tendons were dissected free from the surrounding tissue. Tendon stability may be obtained by passing two sterile 22G needles transversely through the tendon ends inserted into AT stump, about 5 cm apart from each.
20
Figure 6: The arrow shows the site for Achilles tendon hemi-transection which involved 1/3 -to- 1/2 of its full thickness.
Figure 7: The hemi-stranected Achilles tendon was sutured by locking-loop suture pattern, using 3-0 nylon suture material.
21
3.2.4. Immobilization techniques for CG and TG Postoperatively (P.O.) the right pelvic limb in CG dogs was immobilized in a semi-extension position for 3weeks using a full cast either with fiberglass cast, or plaster of Pares (POP). The limb was first padded with a moderate layer of cotton from above the femuro-tibial joint down to above the digits, and wrapped with a moderately tight layer of bandage. Finally, either the fiberglass bandage, or the POP was applied to immobilize the hock.While in TG, the limb was immobilized in a semi-extension position for 3 weeks using a unilateral external fixator device (EFD). The devise was composed of: Schanz pin inserted into the bone, connecting rods and clamps (Tomlinson and Moore, 2008). The procedure for the device application was as follows: 1. Two proximal (above the hock) and distal (below the hock) fixation 2.5 mm Schanz pins were driven through small skin incisions into the tibia and metatarsal bones at appropriate angles (Fig. 8A and 8B). 2. Connecting clamps and fiber carbon bars were slid onto the end pins. 3. The connecting bar was positioned far enough from the dog’s pelvic limb to avoid damage to the skin and muscles due to postoperative swelling formation. 4. At a semiextended hock position, the connecting clamps were finally tightened. At the end of the 3rd. P.O. week, the applied EFD in TG was removed and the dogs were allowed for free walking for one hour daily.
3.3. Intermittent pneumatic compression (IPC) treatment IPC schedules: Seventy two hours following AT hemi- transection, the dogs at TG received a daily intermittent pneumatic compression (IPC) treatment for 45 minutes/day for the subsequent postoperative days. The dogs which remained alive for one week received only four days IPC treatment and those remaining for two weeks received eleven days treated with IPC, while those which remained for 28 days received twenty five days IPC treatment. The IPC session treatment was conducted by Rossmax medical device for blood pressure monitor. The device is composed of two 22
parts; digital blood pressure monitor (the pressure can be set between 40 and 120 mmHg), and the tourniquet cuff. The treated dogs were sedated with xylazine (0.1 mg/kg, IM).
a
Figure8 a: The unilateral external fixator devise used for P.O. immobilization of the dog’s limb in a semi extended position.
b
Figure8 b: Through the skin incisions, four 2.5 mm Schanz type self-drilling pins were fixed above and below the hemi-transected tendon.
23
3.4. IPC procedure 1. The tourniquet cuff was wrapped above the hock joint, directly on the treated AT (Fig 9). 2. The cuff was inflated by Rossmax medical device within 12 seconds to 40-60 mmHg (Fig. 10). 3. After about 48 seconds, the cuff was deflated. 4. This procedure was manually controlled for the inflation and deflation which were done in the range of 3 times/5 minutes. The daily IPC sessions were for 45 minutes/day.
3. 5. Clinical follow up for AT hemi-transection All dogs in both CG and TG groups, were followed up clinically in order to determine the occurrence of any P.O. unusual complication during the course of the tendon.
3.6. Histo-pathology examination 1. Euthanasia of dogs The dogs in either CG or TG were euthanized by administration of a mixture of ketamine xylazine, followed by over dosage of halothane, at three schedules; 7, 14, and 28 P.O. days. Three dogs from each group were randomly euthanized. 2. Gross postmortem examination for the repaired AT was performed. 3. Tendon biopsies were collected from the site of operated Achilles tendon, and then were fixed at 10% neutral buffered formalin for 72 hrs. After which the section was prepared routinely according to Tvedten and Willard (2004). 4. The slides were stained with Hematoxyline-Eosin (H&E) to show tendon healing microscopically in each group.
24
Figure 9: IPC is applied directly above the repaired Achilles tendon in one of the dogs of the treatment group.
Figure 10: IPC device shows12 mmHg pressure applied to the repaired Achilles tendon in one of the treatment group dogs.
.
25
Chapter Four
Results
4.1. Anesthesia All the eighteen dogs entered this study, being well tolerated the anesthetic and experimental procedures. The anesthetic protocol produced smooth induction, and provided good analgesia. Maintenance and recovery with the mixture were without complications. The main side-effects were the profound bradycardia and some cyanosis due to respiratory rate decreases. But, these were not serious, because the dogs were premeditated with atropine sulfate.
4.2. Postoperative fixation of hock Joint Post AT repair, fixation of dog's tarsal joints was successfully performed either by the external fixator devise (EFD), or plaster of Paris (POP) and the tarsus was fixed in a semi extension position. Both fixations were useful for restriction of AT movement during the three P.O. weeks in either CG or TG. On the 2nd P.O. day, the EFD in one dog (No. 4; TG) was broken, because the upper pins fixed to tibia dropped when the dog's leg was caught in a wire cage wall leading to partial rupture of the sutured AT. The ruptured AT remained without repair. It healed, but a gap was felt during palpation. Generally speaking, most of the TG dogs tolerated well the EFD applied to their right pelvic limb to lock their hock joints, but one aggressive and none cooperative dog (No. 6 TG), which in the short-term, faced a minor complication on its operated limb during the early P.O. phase. The complication was in the form of slight swelling and purulent exudates from the site of pin fixation which required reimplantation and fixation of the lose pins under general anesthesia, daily wound cleaning and local and systemic antibiotic therapy. 26
Another dog in the same TG group (No. 9) had an open traumatic lacerative injury on its digits caused by the wires of the fence that cover the wall of the home cage. While, on the other hand, the plaster cast used to fix the stifle joint of the CT dogs was more reliable and in none of the dogs went off from its place for the whole three P.O. weeks. After removal of the immobilizers, both groups, on the end of the 3rdP.O. week the dogs were left to walk free inside their cages and were given an hour daily exercise out of their cages. During which, the dogs showed only a slight walking restriction during the first 24 -to- 36 hours. Then, they showed normal gait walking, running and jumping.
4. 3. Achilles tendon tenorrhaphy The suture material used in the tenorrhaphy (tenosuture) of the Achilles tendons in this study was nylon 3/0 (USP scale), with the Kessler suture pattern.
4. 4. Postoperative clinical signs The gross results, regardless of IPC treatment, during the first week, were sequentially similar in both groups. All had good dexterity (cleavers) and intact resilient skin. No risks like: bleeding, infection, skin loss, post-traumatic and postoperative swelling, distortion and pitting edema. Clinically, all cardinal signs of acute inflammation were seen during the 48 to 72 hours P.O. but, none of the dogs showed systemic reactions and local inflammatory complications, i.e., rupture of tendon suture (except in one; dog no. 4 in TG), severe exudates, and acute signs of pain. During the 48-72 P.O. hours, animals were not able to put weight on the limb. But, later on these signs reduced dramatically and the dogs were putting weight of bearing on the limb at a mean day time ranging between the 5th to- 7th (5± 2.4) P.O. days. On the 14th and 28th P.O. days, in both groups, there were proper healing, complete weight bearing, no gap formation at the site of suturing, and the AT blood vessels were neither pulsating, nor engorged. On manipulation, no significant adhesions between thetendon and the skin and no thickening of the tendon along its length were found (Figure 11). 27
A
B
Figure 11: The image A shows proper healing of the Achilles tendon on the 28th day P.O. in one of the patients in the treatment group, while B shows perfect healing, no regional swelling or gap formation, along the course of the operated tendon.
4. 5. Gross Achilles tendon Findings On the 7th P.O. day, Achilles tendon necropsy, regardless of dogs, showed little significant difference between treatment and control groups. The repaired tendons generally appeared inflamed along their lengths, congested, irregular in shape and lose their usual glistening luster. The regional blood vessels surrounding the Achilles tendon for the CG dog were engorged (Figure 12). While for the TG dogs, the engorged blood vessels still remain and the site of hemi-transected AT increased in diameter (Figure13). On the 14th P.O. day, most of the CG dogs showed a gross thickening along the length of the repaired AT. At the Post-Mortem, the Achilles tendons were hyperemic and adhered to the surrounding tissues, causing some restriction in limb movement (figure 14 A and B), although tendon healing was not adversely affected. While in TG, the repaired Achilles tendons were grossly seen much uniform in shape, not adhered to its surrounding tissues, and with minimum thickness in comparison to CG (figure 15).
28
B
A
Figure12: Post-Mortem for a dog from control group on the 7th P.O. day. A shows engorgement of the regional blood vessels and B shows thickening of the Achilles tendon at the hemi-transacted region.
A
B
Figure13: Post-Mortem for a dog in the treatment group on the 7thP.O. days, i.e., 4 days after application of IPC treatment. A shows engorgement of regional blood vessels and B shows more thickening of hemi-transcected Achilles tendon.
29
A
B
Figure14: Dog No. 11 from the control group on the 14th P.O. day. The image A shows gross Achilles tendon distortion that was causing partial restriction in limb movement and in B adhesions were seen between the tendon and its surrounding tissues (green arrow).
B A
Figure15: A photograph showing the gross Achilles tendon for a dog (No. 9) in the treatment group on the 14th P.O. day. In A, the repaired Achilles tendon is seen cordlike and uniform in shape and B shows absence of adhesions with its surrounding tissues and that the regional blood vessels were not engorged. On the 28th P.O. day with the CG dogs, the healed AT grossly appeared thick and distorted in shape (Figure16), while, on P.M., although they were healed, severely thickened and adhered to their surrounding tissues (Fig. 17).
30
Figure 16: The gross Achilles tendon in the dog of the control group on the 28th P.O. day healed, but it was not uniform in shape and appeared thicker in the middle with a large scar tissue.
Figure 17: Post-Mortem for a dog from the control group on the 28th P.O. day shows the repaired Achilles tendon severely thickened and adhered to the surrounding tissues. On the other hand, grossly the TG dogs in the 28th P.O. days showed a much uniform and cord-like Achilles tendon. 31
The operated right limbs were normal in shape and movement without overstraining of the thigh muscle or extensive hock flexion (figure 18, 19).
Healing of pin places
Figure 18: Image of the dog of the treatment group on the 28th P.O. day showing the repaired Achilles tendon uniform and cord-like in shape. The limb appears normal without overstraining of the tissue or extensive hock flexion.
Figure 19: Post-Mortem, the repaired Achilles tendon from the treatment group on the 28th day P.O. was healed properly and appeared cord-like in shape without adhesions to its surrounding tissues.
32
4. 6. Pathology for Achilles tendon healing The main histo-pathological differences between the control (CG) and treatment (TG) groups (their subgroups) on the dog's Achilles tendon sections stained with Hematoxylin and Eosin (H&E) at the 7th , 14th, and 28th P.O. days were in: fibers structure and
orientation;
concentration of
cellular
elements (tendocytes);
neovascularization; and enhanced fibroblast formation.
4.6.1. The 7th P.O. days Healing for both groups was somewhat similar. Whereby, congested blood capillary with slight hemorrhage and heavy infiltration with inflammatory cells, mainly neutrophiles. On this week, in both groups, irregular arrangement of collagen fibers, which was type I, with the presence of newly formed immature fibroblasts in different structure with few fibrin depositions was seen (Figure 20). In the same session in the TG, after daily application of IPC, which was only for four days, new blood capillary and the migration of more inflammatory cells to the injured site, were observed. Proliferation of immature fibroblasts with different stages was seen round, spindle, and oval (Figure 21).
33
Figure 20: Amicrograph of dog´s Achilles tendon of the control group on the 7th P.O. day. The red arrow shows congested blood vessels, the green arrow shows heavy infiltration of inflammatory cells and the blue arrow shows immature fibroblasts. (H&E, X40).
Figure 21: A micrograph of repairing Achilles tendon from the treatment group on the 7th day. The red arrow shows congested blood vessels, the green arrow shows heavy infiltration of inflammatory cells, the orange arrow shows immature fibroblasts and blue arrow shows collagen fibers less wavy in shape. (H&E, X20).
34
4.6.2. The 14th P.O. day On the 14th P.O. day, the healing process (proliferation stage) for the hemitransected Achilles tendon specimens in CG were the formed tissue remained more immature and infiltrated with tenocytes that were flat in shape with a clearly visible elongated fibroblast nucleus. Also a significant increase was seen in the number of fibroblasts in various stages of development, which were oval and elongated shape, and hemorrhagic area between collagen fibers with newly formed blood vessels (Figures 22 ).While, in the samples for the same period on the 14th day in TG better organized collagen fibers were seen, as the tissue looked denser with fewer elastic fibers, showed a great number of newly formed blood vessels and an increased cellularity especially at the edge of the tenotomy (Figure 23)
35
Figure 22: A micrograph of repairing Achilles tendon on the 14th day in the control group. The red arrow shows new blood vessels, the green arrow shows concentration of immature tenocytes that were flat in shape with a clearly visible elongated fibrocyte nuclei and the blue arrow shows the oval and elongated shapes of fibroblast cells in various developmental stages. (H&E x 20)
Figure 23: A micrograph showing repairing Achilles tendon on the 14th, day in the treatment group. The blue arrow shows fibrous connective tissue formation, the red arrow shows collagen fiber concentrated and infiltrated with mononuclear cells (tenocyte) and the green arrow shows blood vessels. (H&E X 50). 36
4.6.3. The 28th P.O. day At the last session (28th days) in CG dogs, the tendons were seen almost healed, but still some cellular activity was observed in the tissues. The number of vessels largely decreased, indicating progress of the healing process. The proliferation of mature fibrous connective tissue consists of few spindle shaped fibroblast cells with dense collagen fibers, which have discontinued wavy appearance resembling rough scar tissue (Figure 24). While in treatment group the collagen bundles were arranged parallel and have a slight wave, slender, elongated fibroblast cell nuclei. The concentration of cellular elements with a large amount of collagen maturing accompanied by a large amount of granulation tissue enhanced neovascularization and presence of high numbers of fibroblast cells because of IPC treatment (figure 25).
37
Figure 24: A micrograph of repairing Achilles tendon on the 28thday in the control group. The green arrow shows disncontinous wavy appearance resembling rough scar tissue and the blue arrow shows proliferation of mature fibrous connective tissue consisting of few spindle shapes fibroblasts. (H&E X 20).
Figure 25: A micrograph of repairing Achilles tendon from the treatment groupon the 28th P.O. day. The green arrow shows progress in the development of well organized and the blue arrow shows homogenous pattern of collagen fibers. (H&E X20). 38
Chapter Five
Discussion
5.1. Anesthesia
Anesthesia in dogs has several potential risks, and this is perfectly natural. The risks of general anesthesia are usually greater than those of the surgery itself. All patients (18 dogs) in this study were scheduled to undergo a surgical procedure of anesthesia and they had ability to tolerate it. Atropine sulfate (0.02 mg/kg) was used to protect the patient's heart from the adverse bradycardial effect of xylazine. The combined mixture of (Ketamine HCl 100 mg/ml) at dose 10 mg/kg body weight with 5mg/kg of Xylazine 2% according to (Mckelvey and Hollingshead, 2003), induced an excellent, smooth and safe anesthesia within 3-5 minutes, that facilitates handling dogs and aids in conducting diagnostic and therapeutic procedures (Clark, 2001). The dogs were awake and were able to stand on their own, and fully recovered within 4 hours, and no one complicated procedures longer.
5.2. Achilles Tendon tenorrhaphy Tendons are sutured to re-approximate the ends to prevent gap formation, permit glide function and minimal adhesions in order to permit healing. The healthy ends of the tendon were attached back together in a sufficient tissue bed by using locking loop technique. In this study during suturing, the tendon was held by hand, fixed with two sterile needles 22 G and squeezed by fingers bluntly from surrounding tissue instead of using instruments because the instrument handling of a tendon will traumatize the delicate fibroblasts covering of the tendon known as epitenon. Gourley and Vasseure, (2007) mentioned that the mere passages of a suture needle 39
through a tendon produced sufficient trauma to cause local adhesions. Additionally the tendon kept wet to prevent fibrosis, adhesions and poor gliding. Although the strength of suture material contributes to the overall strength of the tendon repair, the technique utilized is more important. In this study, Achilles tendon group sutured with nylon showed better tissue organization, and also the collagen fiber arranged in the load direction. It is presumed that suture material elasticity allows interaction between the suture material and tissues, which results in the greater elasticity of scar tissue during healing. The hemi-transected Achilles tendons were successfully accomplished in all of the cases using the modified Kessler suture technique and none of the repaired tendons failed at the surgical site. All dogs clinically from the 3rd to the 28th P.O. days did not showed potential complications, including rupture, because they could be avoided with appropriate three weeks immobilization cast, good postoperative care, and a daily one- hour course of exercise. Wong et al., (2006) showed that the process of tendon suturing causes cell death directly. Cell death, inflammation and extracellular matrix break down are occurring most dramatically in the areas of highest stress in repairs, i.e., around the locking knot. As noticed in this study none of the sutured Achilles tendon stitches, in both groups, ruptured. So, it was an adequate choice of suture material, as tendons need a great deal longer to heal, and during that healing period they need the mechanical support of non-absorbable suture material. Nylon thread tends to be springy and requires more knots to prevent untying (Kreszinger et al., 2011), The main characteristic of nylon, as opposed to wire and polypropylene, is elasticity also Meutstege, (1993) believed that nylon is an ideal material for tendon suturing due to the minimal tissue reaction. Traditional tendon sutures employed two core strands bridging the gap between the tendon ends. When the internal suture is present and tension is applied during suture, prolonged inflammation occurs at the sites of placed suture, which potentially stimulates adhesion formation. Moller, 2001 mentioned that common reason for unsuccessful tenorrahphy is the suture material cutting in along collagen fibers. Wada et al., (2001) also came to the conclusion that the elastic material allows more pulling strength for tendons. They chose drawing ability as a measure of deformability to form a complete view of scar tissue quality. The Achilles tendon is naturally exposed to frequent stretch loads. Unless the scar tissue is adequately resistant to deformations, a loss of structure will occur and permanent deformations can result, which affects the Achilles tendon function (Kudding, 2010). It has been demonstrated that more strands of suture 40
and greater caliber of suture both increase the strength of the repair and hence mitigate against rupture (Strick, 2000). However, it has also been shown that more suture material on the outside of the tendon encourages adhesion formation (Zeo et al., 2001). Because of the poor blood supply to the tendons, tendons were inert and could not heal without extrinsic adhesions. These adhesions led to a stronger repair without the ability to glide (Daniel, 2004). Montgomery and Fitch (2003) mentioned that the use of strong, inelastic suture is of primary importance, so the strength of the sutured repair must be sufficient to prevent failure and gap prevention to optimize healing by promoting primary healing. Also, the suture should not obstruct the local blood-supply and provoke diastasis of the tendon ends (Fossum et al., 2004).
5.3. Postoperative fixation of hock joint Dog leg injuries happen more commonly than just about any other type of dog injury which include injuries to the skin, muscle, ligaments, tendons, bones, or paw. Dogs are very good at hiding injuries, a behavior that stems from an atavistic survival mechanism. As a result, it is seldom realized that dogs are in pain until the damage is serious. It is worth noting that dog leg injuries happen very easily because less force is required to cause such injuries. Rupture of the AT at the hock joint can be caused by sudden and extreme flexion of the hock, most often severed in dog fights and car accidents. Treatment of Achilles tendon injuries including severing or rupture can broadly be classified into operative (open or percutaneous) and non-operative (cast immobilization or functional bracing), (McComis et al., 1997). When compared with immobilization, surgery provides less chance that the tendon will rupture again and offers a shorter recovery period (Hamish and Stevan, 2006). In this study, repair was by both surgery and fixation at the same time. Lister et al., (1997) reported the use of early passive mobilization to induce relative motion between the tendon and paratenon and ultimately reduce restrictive adhesions. Without mobilization rupture is less likely, but adhesions are more likely to form. Hence focus on tendon repair is on having a high repair strength in order to survive early active mobilization and avoid both rupture and adhesion formation (Lister et al, 1997).
41
The repaired ones were protected by immobilization of the hock joint to prevent early excessive weight-bearing postoperatively for proper tendon healing. Fixation was placed on the tarsus at a semi normal slight extension position. Montgomery and Fitch, (2003) demonstrated the importance of immediate stress on the repaired tendon by the release of weight bearing and muscle contraction. This study has shown that upon the early mobilization tendons exhibited improved strength throughout healing, and reduced adhesion formation and excellent weight bearing. Furthermore, when immobilization was combined with IPC, better healing, minimal joint stiffness and lesser adhesions were obtained in the TG dogs. Complications such as rupture, adhesions, and joint stiffness may occur after Achilles tendon repair (Hameshy and Stevan, 2006). Re-rupture of a repaired tendon is due to unplanned high loads that exceed the tolerance of the repaired tendon. It may also occur from early excessive exercise or unexpected pelvic limb motion, such as suddenly falling. With immobilization, the strength of tendon repair has been shown to decrease significantly within the first three weeks of healing (Hitchcock et al., 1987).Two different methods of immobilization were used for the operated dog's right pelvic limb during the 3P.O. Week, for both groups. External fixation devices (EFD) was used for the TG and plaster of Paris (POP) was used for the CG. The EFD was applied to stabilize the hock joint whilst soft tissue healing took place, allowing at the same time the wounds to receive daily topical treatment and daily application of the IPC. The EFD frame was applied to the lateral aspect of the right pelvic limb, and worked quite well in most of the 9TG dogs. During post-operative management, primarily in the early stages of healing, limb immobilization is essential (Millis et al., 2004). Movement can produce a significant space between the tendon ends, decreasing blood supply and increasing fibrosis, which can compromise healing and final functional outcome (Clark, 2001). Early mobilization of an injured tendon reduces the proliferation of fibrous tissue and reduces the formation of adhesions between the tendon and its sheath. This study agrees with the latter and others (Fossum et al., 2004). The repaired Achilles tendons in the patients, particularly the TG dogs, presumed minimal adhesions (confirmed grossly) and reduced proliferation of fibrous tissue (confirmed histopathologically). Some dogs may always have some degree of lameness, abnormal limb position, or abnormal toe nail position (Drisko, 2012). By the 3rd week remodeling phase started where the injured tendons in the dog s´ leg were weight overloading of AT by removing of the immobilizations from their limbs because in 42
this phase it requires a slow but continuous loading of the tendon over time (Clark, 2001; Oakes, 2003).
5.4. Effect of IPC treatment
IPC is used as an effective treatment for a variety of circulatory disorders in humans (Schizas, et al., 2010). Its uses for venous thrombo embolism prophylaxis and treatment of lymphedema is well established (Urbankova et al., 2005). It also enhances Achilles tendon early repair by up regulating its local essential metabolites. This metabolic response can, during tendon healing with plaster cast immobilization, be promoted by adjuvant IPC (Greve et al, 2012). IPC also improves walking distance in patients with intermittent claudication and is effective in patients with critical limb ischemia (Turpie et al., 2007). In this study, promote in tendon healing by the IPC procedure was successfully produced by means of the human blood pressure device. The lower pressures (60 mmHg) together with shorter inflation and deflation times (1 min/cycle) appeared to be more efficient than higher pressures and long inflation/deflation times. The IPC device compresses the limb. One second later, the gastrocnemous muscle is compressed. As a result, the hock and common calceneal veins are almost completely emptied. In return, the arterial blood is more easily pushed down to the toes and blood-deprived tissues. Because of this mechanism, blood flow to the skin of the limb can be tripled (Nicolaides et al., 1997). A second mechanism of action that accounts for the large blood flow increase involves the endothelium (cells that comprise the lining of all blood vessels). Endothelial cells release important biochemical factors, such as nitric oxide (NO) that helps circulate the blood. Since these biochemical factors dissipate after approximately 12 seconds, the device repeats the compressed sequence one time per minute. This means that in a 45-minutes session, the patient’s arteries will be expanded. The pelvic limb will suffer cramp more than at 60 mmHg pressure. Lower pressures together with shorter inflation and deflation times appear to be more efficient than higher pressures and long inflation/deflation times (Eze et al., 1996). The cuff was wrapped on the injured Achilles tendon, when intermittently inflated and deflated physically, enhanced local blood circulations, which were partially reduced by limb immobilization. Kraemer et al, (2009) reported that Achilles tendon suture causes an acute loss of capillary 43
perfusion and increases post capillary venous filling pressures indicating venous stasis. In this study, it is believed that the use of IPC treatment overcomes this effect. It enhanced tendon repair by stimulating local blood flow, encouraging the proliferation of fibroblasts and endothelial cells at the site of tendon injury. Immobilization of the CG dog's limb with the POP was practical, cheap and free of complications, while, the EFD used for TG dogs was accompanied with malfunction due mainly to pin loss. Additionally, the site for the pin fixation of the tibia and metatarsal bones was not free from infection. However, the difference in the type of immobilization between both groups did not affect the rate or final functional outcome for AT healing. On the second week, all had the ability to actively bend their toes because their tendons were strong and also have a long gliding excursion through the hock and toes. However, tendons have a fairly balanced combination of brittleness (resistance to force without having a plastic range) and ductility (capacity for deformation without failure). The positive early effect of treatment with the IPC was positively reflected on the differences in the gross and histological healing features for the AT seen between the CG and TG. During the treatment sessions, the Achilles tendons for the CG grossly appeared pale, lightly swelled and thick along its length. This was believed to be attributed mainly to the restriction in regional blood supply produced by the POP immobilizer on the cast right pelvic limbs. While, the Achilles tendons in the TG looked thicker in diameter and the surrounding blood vessels were engorged with blood, an adequate blood supply is necessary for tendon healing as reported by workers (Koth and Sewell 1988; Gebauer, 2007; Lewis and Quitkin, 2003; Lansdaal, 2007). These gross changes in the TG were obtained by cuff which pressurizes the tissues in the limb, thereby forcing fluids, such as blood and lymph out of the pressurized area. A short time later, the pressure is reduced, allowing increased blood flow back into the limb. IPC increased the rate of healing by improving arterial blood flow documented both in the popliteal artery and by foot skin perfusion and increased transcutaneous oxygen measurements (Urbankova, 2005). Additionally, IPC treatment could counteract morphological deficits caused by immobilization by enhancing soft tissue repair, as one week of immobilization was already found to be detrimental for muscle tissue (Hurme et al., 1990). IPC promotes tissue repair by stimulating tissue perfusion and nerve ingrowth as well as accelerating both fibroblast proliferation, collagen reorganization. 44
5.5. Histological finding of the repaired AT Presence of minor differences in the histological parameters during the 7th P.O. day between the two groups in the structure of the inflammatory cells could be attributed to the short period of IPC treatment, where, the dogs in the TG only received the IPC treatment for 4 days, which was not enough to promote tendon healing. The tissue for both groups showed the irregular arrangement of collagen fibers focused in different directions. The injured tendons were congested and hemorrhagic and heavily infiltrated with inflammatory cells. After a few days, the proliferative phase begins. Synthesis of type-III collagen peaks during this stage and lasts for a few weeks. Thickness of the tendon along its length due to increase in water and extracellular matrix ECM, these histological changes are the same as reported by Fenwick et al., 2002 that mentioned that water content and glycosaminoglycan concentrations remain high during this stage. On the 14th P.O. day, it seems that IPC treatment positively strengthened the healing phases for the Achilles tendon as observed by the elevated numbers of blood vessel capillaries. Fibroblast density at all time points significantly increased in the IPC group. In the TG tissue specimens, a higher amount of fibroblast cells was seen, compared to CG tendons. On the 28th P.O. day, IPC treated tendons increased tissue organization. This was confirmed by decreased fibroblast with the development of a much more organized and homogenous pattern of collagen fibers which has a smooth appearance resembling semi normally tendons. The number of vessels enormously decreased, indicating the progress of the healing process, and fibroblast density returned to normal. From all the above, at this session the TG tendons displayed a higher degree of organized parallel collagen fibers, a sign of increased maturation. While, on the other hand, tendons for CG dogs seem almost healed, but lacking a normal tendon texture, due to laxity of the collagen fibers which were showing discontinuous and disorganized wavy matures fibrous connective tissue. Misalignment of collagens with each other was observed. Tendons need approximately one year after surgery to heal completely (Drisko, 2012). Additionally, fatty cells deposit between the collagen fibers caused disrupted alignment, fiber separation and thinning. Accumulations of ground substance deposits lead to delaying in healing (Sharma and Maffulli, 2005). 45
Conclusion 1. IPC proved to be an effective modality for the stimulation and repair of injured Achilles tendons in dogs. 2. When IPC is combined with a short period of immobilization, it enhances Achilles tendon healing. 3. IPC markedly reduced and /or prevented occurrence of adhesions.
Future researches 1. Comparing the effect of presurgical immobilization with postsurgical immobilization on healing of the injured Achilles tendon. 2. Studying the effect of allograft, autograft, xenograft, synthetic tissue, or biologic adjuncts in the treatment of Achilles tendon injuries. 3. Studying the use of antithrombotic treatment on the healing of immobilized Achilles tendon in injuries. 4. Use of IPC on other tissues (nerve ingrowths; fractured bone; soft tissue) to enhance healing.
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ﺗﺄﺛﯿﺮ أﺳﺘﺨﺪام اﻟﻀﻐﻂ اﻟﮭﻮاﺋﻲ اﻟﻤﺘﻘﻄﻊ )(IPC ﻋﻠﻰ اﻟﺘﺌﺎم اﻟﻘﻄﻊ اﻟﻨﺼﻔﻲ ﻟﻮﺗﺮ اﻟﻌﺮﻗﻮب اﻟﻤﺜﺒﺖ ﻓﻲ اﻟﻜﻼب رﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ اﻟﻰ ﻣﺠﻠﺲ ﻛﻠﯿﺔ اﻟﻄﺐ اﻟﺒﯿﻄﺮﯨﻔﻲ ﺟﺎﻣﻌﺔ اﻟﺴﻠﯿﻤﺎﻧﯿﺔ ﻛﺠﺰء ﻣﻦ ﻣﺘﻄﻠﺒﺎت ﻧﯿﻞ ﺷﮭﺎدة ﻣﺎﺟﺴﺘﺮ ﻋﻠﻮم ﻓﻲ اﻟﻄﺐ واﻟﺠﺮاﺣﺔ اﻟﺒﯿﻄﺮﯾﺔ ﻣﻦ ﻗﺒﻞ دﯾﺪن ﻣﺤﻤﺪﺗﻘﻰ ﻣﺤﻤﺪاﻣﯿﻦ ﺑﻜﺎﻟﻮرﯾﻮس ﻓﻲ اﻟﻄﺐ واﻟﺠﺮاﺣﺔ اﻟﺒﯿﻄﺮﯾﺔ ),(2004 دﺑﻠﻮم ﻋﺎﻟﻰ ) (2011ﻓﻰ اﻟﺠﺮاﺣﺔ اﻟﺒﯿﻄﺮﯾﺔ, ﺟﺎﻣﻌﺔ اﻟﺴﻠﯿﻤﺎﻧﯿﺔ ﺑﺎﺷﺮاف اﻟﺪﻛﺘﻮر ﺑﮭﺠﺖ ﻃﯿﻔﻮر ﻋﺒﺎس أﺳﺘﺎذ
ﺗﻤﻮز 2014ﻣﺮﻣﻀﺎن 1435ه
AT IPC IPC IPCAT 21 18 mg/kg10(2 mg/kg) Ketamine hydrochlorideXylazine 3 30 CG 9 EFD TG 9 IPC 45 72 TG IPC IPC ii
IPC AT
iii
(IPC) 20112004 و
20142714
