EVALUATION OF SOME CYTOKINES IN UNINJURED AND CUTANEOUSLY INJURED NIGERIAN INDIGENOUS DOGS

Abstract

Twelve apparently healthy Nigerian indigenous dogs (NIDs) (6 males and 6 females) within the age range of 9 – 13 months and weight range of 8 – 12 kilograms were assigned to experimental (injured dogs) and control (uninjured dogs) groups to determine the levels of some cytokines and blood cell counts in cutaneously injured and uninjuredNIDs at the Ahmadu Bello University Veterinary Teaching Hospital, Zaria. Both circulatory (serum) and wound fluid cytokines were studied in experimental group, while those in control group were used to study circulatory cytokines. Blood samples from each dog were collected via cephalic venopuncture at start (pre-operative), 12, 36, 60, 156 and 324 hours post operative and refrigerated in plain (for harvesting of serum) and anticoagulant-containing (for blood cell counts) sample bottles. Serum and wound fluid collected at 12, 36, 60 and 156 hours were stored at -20 ^oC for cytokine assay. Canine specific ELISA kits were used to assay for concentrations of cytokines [interleukin (IL) -6, -8, -10 and tumour necrosis factor (TNF) – α] as described in Abcam product protocol (ABCAM^® UK, 2015). Results revealed peak concentrations of IL-6 in wound fluid (1.33. ± 0.33 ng/mL) and serum (1.13 ± 0.74 ng/mL) at 12 h, post-operation were higher (P < 0.01) than the control (0.30 ± 0.05 ng/mL). There was no significant (P > 0.05) difference in circulatory IL-8 levels between experimental and control group. Circulatory IL-10 concentration (1.67 ± 0.33 ng/mL) was significantly (P 001) higher than those in wound fluid (0.87 ± 0.03 ng/mL) of experimental and control (0.94 ± 0.17 ng/mL) groups at 156 h. The concentrations of TNF-α in wound fluid (130.22 ± 35.15 pg/mL) was significantly (P < 0.05) higher than in serum (44.50 15.92 pg/mL) of the experimental group at 36 h, while the serum (116.17 ± 60.27 pg/mL) was higher than the control (29.79 ± 7.47 pg/mL) values at 60 h post-operation. Mean   haematocrit   of   0.354   ±  0.01   recorded   at   12   hours   post-operation   in   the experimental group was lower than the corresponding values of 0.364 ± 0.00 recorded in the control group. The total Leucocyte counts remained within the normal limits with minimal changes in differential cellular activitiesof theexperimental and control group.Significant correlations of serum IL-6 with wound fluid IL-6 (r = 0.827; P < 0.05), wound fluid IL-6 with HCT (r = – 0.893; P < 0.05) and RBC (r = – 0.923; P <0.01), wound fluid IL-6 with monocytes (r = 0.818; P < 0.05), serum IL-10 with lymphocytes (r = 0.846; P < 0.05) and wound fluid TNF-α with monocytes (r = 0.824; P 05) were observed in injured NIDs. Interleukin (IL) -6, -8, -10 and TNF-α of the experimental group were significantly (P < 0.05) higher than those of the control group of NIDs. Significant correlations were observed with IL -6, -10 and TNF-α and red blood cells, lymphocytes and monocytes of the NIDs within a range of r = 0.818 – 0.923. In conclusion, maximum mean concentrations of interleukin (IL) -6, -8, -10 and TNF – α detected at 450 nm from uninjured NIDs were 0.30 ± 0.05 ng/mL, 383.16 ± 157.57 pg/mL, 0.94 ± 0.17 ng/mL and 47.06 ± 14.34 pg/mL, respectively.

CHAPTER ONE

INTRODUCTION

1.1         Preamble

Cytokines are low molecular-weight proteins (5-20 kDa) that are released by epithelial cells, endothelial cells, fibroblasts, activated lymphocytes, macrophages and monocytes and are important in cell signalling (Tracey et al., 1987; Dinarello, 1994; Wang and Tracey, 1999; Horst, 2013;Orzechowski, 2017). They mediate the metabolic and physiologic responses to injury and infection by binding to specific receptors, enabling immune cells communication, differentiation, proliferation and other activity which trigger inflammatory response to sites of injury and infection thereby enhancing wound healing (Haichao et al., 2000; Horst, 2013; Orzechowski, 2017). Cutaneous injuries are wounds which may be surgically induced or occur naturally. A wound is a traumatic separation/disruption of continuity of the skin, mucus membrane or an organ surface and it is one of the most frequent reasons for seeking medical attention (Hassan et al., 2002; Kauvar and Wade, 2005; Ahmed et al., 2013; Borena et al., 2015). Wound is also defined as a loss or breakage of cellular and anatomic or functional continuity of living tissue following road traffic accidents, falls, cuts from sharp objects such as corrugated iron sheets and barbed wires, assault/acts of violence by individuals and surgery amongst others (Milner, 2008; Kokane et al., 2009; Karen and Spencer 2012). Injury to the skin initiates series of events, which finally results to at least a partial reconstruction of the wounded tissue (Martin, 1997; Auf-dem-Keller et al., 2006; Turkel et al., 2014). Wounds, when left untreated may become complicated, leading to varying degrees of contamination, deformities and ultimately may result in the death of the animal (Amber and Swaim, 1984; Hassan et al., 2003; Namas et al., 2009). When a wound is infected, it contains replicating micro-organisms which elicit a host response causing injury to the host. Etiologically, wounds are categorized into acute or chronic forms comprising surgical wounds, burns, lacerations and puncture wounds (Kokane et al., 2009). In an acute wound, infection is met by a rapid inflammatory response initiated by complement fixation and an innate immune response followed by the release of cytokines and growth factors (Dow et al., 1999).

Cytokines are generally classified into Pro- and Anti-inflammatory cytokines, with the pro-inflammatory cytokines comprising of the tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) and anti-inflammatory cytokines which includes interleukin-10 (IL-10) amongst others. Others forms of cytokines include interferons and lymphokines (Blackwell and Christman,1996; McBride et al., 1996; Lin et al., 2000; Bown et al., 2001; Chachkhiani et al., 2005). Wound healing is a complicated and very highly regulated pathway that varies in its processes longitudinally, temporally and spatially and involves a few phases (the main phases: Inflammation, Proliferation involving debridement and repair, and Maturation) which consumes vast resources (Hassanet al., 2002; Grazul-Bilska et al., 2003; Nguyen et al., 2009; Guo and Dipiero, 2010). The inflammatory cascade produces vasodilation and a significant increase in blood flow to the injured site, thus increased cytokine response facilitating the breakdown of devitalised tissues while promoting the formation and preserving healthy granulation tissues with resultant restoration of tissue architecture and function (Robinson, 1993; Reddy et al., 2008). The various pro- and anti-inflammatory cytokine profile can be measured in serum and wound fluids by quantitative enzyme linked immunosorbent assay (ELISA) using the cytokine canine ELISA kits with preset minimum detectable levels of the cytokines by the manufacturers (Zuhoor, 2012; John et al., 2013). Pro-inflammatory cytokines such as tumour necrosis factor (TNF) and interleukin 1 (IL-1) are released by activated lymphocytes, macrophages and monocytes during infection, and acute inflammation following injury or wound (Tracey et al., 1987; Dinarello, 1994; Wang and Tracey, 1999). Pro-inflammatory cytokines are pyrogenic and can stimulate thermogenesis by directly acting on the hypothalamic centers (Jones, 1993). These cytokines, especially TNF and IL-1 also influence the release of corticotropin, growth hormone (GH), pituitary hormones, prolactin, and macrophage migration inhibitory factor (MMIF), which function in the integration of neuro-endocrine and immune responses to various types of injuries and infections (Yamaguchi et al., 1991; Nash et al., 1992; Bucala, 1996; Fry et al., 1998). Consequent to severe trauma, serum levels of TNF-α, IL-6, and IL-8 are significantly elevated. There appears to be a direct relationship between the extent of pro-inflammatory cytokine release and the severity of injury as well as hospital mortality (Hoch et al., 1993; Carlstedt et al., 1997; Jiang et al., 1997). Anti-inflammatory cytokines such as IL-10, TNF-binding protein, IL-1 receptor antagonist (IL-1ra), and transforming growth factor beta (TGF-β) usually produced through a normal immune response, can inhibit the release of TNF- α and other pro-inflammatory cytokines by interfering with their binding to their respective receptors(Sakic et al., 2009). Similarly, stress hormones such as epinephrine, glucocorticoids, norepinephrine, and α-melanocyte-stimulating hormone also inhibit cytokine production (Sakic et al., 2009). There is a correlation between pro- and anti-inflammatory cytokines triggered by the Proinflammatory cytokines (McBride et al., 1996). Pro-inflammatory cytokines such as TNF-α and IL-1 have short half-lives and system of membrane reservoirs (local control)(McBride et al., 1996).The reservoirs include the compromised vascular endothelium with its associated inflammatory and haemostatic activities which hold the cytokines close to the site of release and facilitate the modification of the host inflammatory response by the anti-inflammatory cytokines (McBride et al., 1996). Although cytokine activities have been stated by several researchers to be advantageous to wound healing, excessive cytokine activities are deleterious(McBride et al., 1996). Some instances are as cited by McBride et al. (1996), where inappropriate synthesis and release of cytokines known as cytokine excess occurs with excessive and/or persistent activation of macrophages and neutrophils following some infectious or non-infectious conditions, burns, haemorrhagic shock, surgery, trauma and ischaemia-reperfusion injuries (Sakic et al., 2009; Zura et al., 2009). If this inflammatory response escapes local control, there will be widespread activation of inflammatory cascades with resultant systemic inflammatory response syndrome (SIRS) (McBride et al., 1996). This SIRS evokes further release of inflammatory cytokines hence, a downward spiral of multi-system organ dysfunction/failure (MSOD/MSOF) and ultimately multiple-system organ dysfunction syndrome (MSODS) such as; kidney glomerular injury, tubular damage, left ventricular dysfunction and vasodilation, acute lungs injury and acute respiratory distress syndrome (ARDS), increased synthesis of hepatic acute phase proteins, inhibition of fibrinolysis with a role in the development of disseminated intravascular coagulation (DIC) and thrombosis (Pagani et al., 1992; Esmon et al., 1994; Pawelec et al., 1995; Kelly and Smith,1997;Oral et al., 1997; Sheeran and Hall,1997; Bown et al., 2001; Kudoh et al., 2001; Shi-long et al., 2016).

1.2         Statement of Research Problem

The Nigerian Indigenous Dog popularly referred to as mongrel are prone to injuries because they are well known for their hunting efficiency, as shepherd dogs, guard dogs at human inhabitants and company to farmers (Olayemi et al., 2009; Olumayowa, 2011). For these and other uses, they are vulnerable to injuries especially cutaneous injuries(Hassan et al., 2003). Often than not, their injuries heal but with others injuries may linger for longer than necessary either because of renewal of the already existing injuries or because of lack of care(Hassan et al., 2003; Swai et al., 2010). Even though it is known that injuries will heal whether aided or unaided, if not they die, the NIDs are exposed to these two sides of healing or healing defects. It is therefore important to have a clean understanding of factors that may assist healing of injuries in the NIDs. Healing of open injuries are further complicated or slowed down by numerous factors as described by Hassan et al. (2003); Benjamin and Christopher (2009); Velnar et al. (2009); Shashi et al. (2013) and Turkel et al. (2014).Effective wound healing process involves a delicate interplay of the various inflammatory cytokines and chemokines, destructive and constructive enzymes, growth factors, extracellular matrices and reactive oxygen species with the various cell types such as red blood cells, keratinocytes, endothelial cells, fibroblasts, platelets, neutrophils, monocytes, macrophages, lymphocytes and nerve cells(Dipietro, 1995; Cummings and Merriam, 2003; Grazul-Bilska, 2003). The continued modifications and changes of the various cytokines, cells and other contributory factors resulting in effective wound healing are thought to vary based on the origin and nature of insult as well as the duration of pathology (Dipietro, 1995; Cummings and Merriam, 2003; Grazul-Bilska et al., 2003). These modifications have been influenced by various systemic and topical agents such as antimicrobials, wound healing ointments and creams though with useful but limite outcomes (Benjamin and Christopher, 2009, Sheela et al., 2012). Over the years, inappropriate responses to injury have been found to be more harmful than the original insult with resultant generalized systemic inflammatory response which damages distant tissues and causes more injury (Tracey, 2002; Van Westerloo et al., 2005; Shi-long et al., 2016). Increased Pro-inflammatory cytokine concentrations in plasma/serum have been demonstrated in humans after major surgeries (McBride et al., 1996; Salo, 1996; Johnston and Webster, 2009) with the magnitude of the cytokine-mediated inflammatory response related to the extent of the surgical insults (McBride et al., 1996). Littleefforts have been made to explore the modulating (upregulation and downregulation) effects of appropriate cytokines to modulate wound healing process and to use the levels of the cytokines expressed in serum to predict disease outcomes (Francisco et al., 1999; Carlstedt et al., 1997; Moseley et al., 2004; Ahmed et al., 2013).

1.3         Justification for the Study

The various forms of traumatic injuries present major challenges to clinicians as they are usually acute in onset and may progress into chronic health problems especially when complicated by infections often resulting in relatively reduced quality of life affecting far beyond the individual victims (Kauvar and Wade, 2005; Namas et al., 2009; Turkel et al., 2014). However, following injuries, survival depends on the individuals’ immune system’s ability to respond and defend the body against further injuries and attack from invading pathogens. The extent of such a response is of critical importance as appropriate responses involving cytokines synthesis and release is a key component of the non-specific (innate) immune system’s response (Tracey, 2002; Van Westerloo et al., 2005). Site, severity of injury as well as nutrition and diet may impact these responses one of which is the inflammatory response. This inflammatory response driven by cytokines and chemokines is partly propagated by damaged tissue-derived products (Damage-associated Molecular Patterns; DAMP’s) which maintain inflammation through pro-inflammatory cytokines release or inhibition by anti-inflammatory cytokines (Namas et al., 2009). Various animal models with traumatic injuries and haemorrhagic shock in dog, mice, pigs, rats, and non-human primates have been utilised in an attempt to move from bench to bedside in understanding the biology of wound healing (Kauvar and Wade, 2005; Namas et al., 2009; Kemin et al., 2014). At the moment, knowledge derived from other studies has been useful in the understanding of cytokine dynamics in other animal species and breeds than injured NIDs (Namas et al., 2009; Zuhoor, 2012; Anonymous², 2015; Bertha et al., 2017). Thus, using the NID in this study, sufficient data when collected would serve as baseline, adding to the worth of knowledge and potential in the development of assessment/monitoring tools in diagnostic, therapeutic and prognostic evaluation and management of cutaneous injuries.

1.4         Aim and Objectives of the Study

1.4.1    Aim

This study aim at evaluating some cytokines in uninjured and cutaneously injured NIDs at the Veterinary Teaching Hospital (VTH), Zaria.

1.4.2     Objectives of the study

To:

1. Determine quantitatively the levels of interleukin (IL) -6, -8, -10 and tumour necrosis factor (TNF) -α in serum of uninjured NIDs.
2. Determine the levels of interleukin (IL) -6, -8, -10 and tumour necrosis factor (TNF) -α expressed in the serum and wound fluid of injured NID

• Evaluate the blood cells (erythrocyte, total and differential leucocyte counts) in uninjured and injured NIDs.

1. Correlate the circulatory levels of IL-6, IL-8, IL-10 and TNF-α in injuredNIDs with each other and with the blood cell counts.

1.5         Statement of Research Hypotheses (H₀)

1. There are no significant differences between the levels of circulatory cytokines of uninjured (control group) and injured (experimental group) NIDs.
2. There are no significant differences between the levels of circulatory cytokines and that in wound secretions of injured NIDs.
3. There are no significant correlations between the various studied cytokines, and the cytokines with the haematologic parameters in injured NIDs.

1.6         Scope

1. Only NIDswere used as study subjects.
2. Matured males and females not above the age of 2years were used.
3. Only Interleukin (IL) -6, -8, -10 and Tumour Necrosis Factor (TNF) –α were assayed
4. Blood samples were collected pre operative (start), 12 h., 36 h., 60 h., 156 h. and 324 h. post operative and sera harvested for evaluation of cytokines.
5. Wound fluids secreted at12 h., 36 h., 60 h., and 156 h. post operation were obtained for Enzyme Linked Immunosorbent assay by a surface flush into sterile containers using normal saline.
6. Basic haematology, urinalysis and serology (blood urea nitrogen, creatinine, glucose, total protein, albumen, sodium ion, potassium ion, hydrogen bicarbonate, chloride ion, calcium ion and phosphate ion) were evaluated to ascertain the healthiness of the NIDs.