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

• 0.001) 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 activities of the experimental 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

• 0.05) 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).