Università di Pisa
Scuola di dottorato in Scienze Agrarie e Veterinarie
Programma in Medicina Veterinaria
Settore Clinica Chirurgica e Ostetrica Veterinaria (07/H5)
Clinica Chirurgica Veterinaria (Vet/09)
STRESS-RELATED BIOMARKERS,
DYSPHORIA AND PAIN IN DOGS
UNDERGOING GENERAL
ANAESTHESIA AND SURGERY
Table of Contents
1-Abstract 4
2-Introduction 7
2.1-Stress Response 8
2.1.1-Perioperative stress 12
2.1.2-Stress and anaesthesia 17
2.2-Consequences of perioperative stress 18
2.2.1-Cardiovascular system 18
2.2.2-Coagulation 19
2.2.3-Pulmonary function 19
2.2.4-Gastrointestinal system 21
2.2.5-Immune function 21
2.3-Fentanyl 22
2.4-Spinal Analgesia 23
2.5-Peripheral Nerve Blocks 24
2.6-Aim of the Study 24
2.7-Hypotheses 25
3-Stress and anaesthetic management 26
3.1-Materials and Methods 27
3.1.1-Animals 27
3.1.2-Anaesthetic Management 28
3.1.3-Intraoperative evaluation 29
3.1.4-Sample collection 29
3.1.5-Statistical analysis 30
3.2-Results 32
3.3-Discussion 35
4-Stress, dysphoria and pain in dogs undergoing stifle
surgery 36
4.1-Materials and Methods 37
4.1.1-Animals 37
4.1.2-Anaesthetic Management 38
4.1.3-Analgesic Protocols 39
4.1.4-Intraoperative evaluation 41
4.1.5-Postoperative evaluation 42
4.1.6-Sample collection 42
4.1.7-Statistical analysis 43
4.2-Results 50
4.3-Discussion 60
5-Stress and Fentanyl 65
5.1-Introduction 66
5.2-Materials and Methods 66
5.2.1-Animals 66
5.2.2-Anaesthetic Management 67
5.2.3-Infusion Protocols 68
5.2.4-Intraoperative evaluation 68
5.2.5-Postoperative evaluation 69
5.2.6-Sample collection 69
5.2.7-Statistical analysis 70
5.3-Results 72
5.4-Discussion 75
6-Conclusions and clinical relevance 76
Introduction
General anaesthesia and surgery have been reported to cause stress in animals. Perioperative stress is associated with increased morbidity. Serum cortisol has been widely used as a stress marker. Dysphoria and pain can cause stress in animals.
The present study evaluates stress response, dysphoria and pain associated with different premedication protocols (study 1), in dogs undergoing stifle surgery receiving different analgesic protocols (study 2), or in association with fentanyl administration in dogs under general anaesthesia (study 3).
Methods
All the studies of this project were performed after Ethical Committee approval.
Study 1: Seventeen healthy dogs belonging to a guide dog for blind school were randomly divided into two
groups on the basis of the premedication (acepromazine 10 µg/kg and methadone 0.1 mg/kg) administration route (intramuscular or intravenous) as part of a standard anaesthetic protocol for non invasive diagnostic procedures. Blood samples were collected on arrival in the hospital (TAR), induction of
general anaesthesia (TINT), extubation (TEXT), and one hour after extubation (TPST). Serum cortisol was
analysed in all samples. Dysphoria was evaluated postoperatively by a blind researcher.
Study 2: Forty-five client-owned healthy dogs were divided into three groups which were randomly
assigned to receive fentanyl VRI (dose adjusted to avoid nociceptive response), spinal anaesthesia (0,05 ml/ kg 0,5% isobaric bupivacaine), or femoral and sciatic nerve block (0,3 ml/kg 0,5% bupivacaine) as part of a standard anaesthetic protocol during stifle surgery. Fifteen healthy dogs undergoing non invasive orthopaedic diagnostic procedures under general anaesthesia were included as control group. Blood samples were collected on arrival in the hospital (TAR), induction of general anaesthesia (TINT), extubation
(TEXT), and one hour after extubation (TPST). Serum cortisol was analysed in all samples. Dysphoria and
pain scores were evaluated postoperatively by a blind researcher.
Study 3: Six healthy purpose dogs were enrolled in this double blinded cross over study and anaesthetised
twice with a wash-out period of 15 days with a standard anaesthetic protocol, and fentanyl (5 µg/kg + 7.5 µg/kg/hr) or NaCl infusion. Blood samples were collected on arrival in the hospital (TAR), induction of
general anaesthesia (TINT), extubation (TEXT), and one hour after extubation (TPST). Serum cortisol was
analysed in all samples. Dysphoria was evaluated postoperatively by a blind researcher.
Results
Study 1: No differences were evinced among the two groups for age, weight, preoperative behaviour score,
postoperative recovery score, glucose or cortisol levels at any time point.
Study 2: In control, spinal and peripheral nerve block groups, serum cortisol and blood glucose were
within normal levels at all time point and did not change after surgery. In the fentanyl group, cortisol and blood glucose levels were significantly higher after surgery (TEXT and TPST) compared to the other 3 groups
and to the values registered before surgery (TAR and TINT). Dysphoria and postoperative pain scores were
significantly higher in fentanyl group compared to the other groups. Fentanyl group required higher doses of opioids postoperatively.
Study 3: No differences were evinced among the two groups for age, weight, preoperative behaviour score,
postoperative recovery score, glucose or cortisol levels at any time point.
Conclusions
Premedication administration route does not modify the stress response in dogs. Spinal anaesthesia and peripheral nerve blocks improve the postoperative pain scores, reduce the occurrence of dysphoria and prevent the increase of cortisol levels compared to fentanyl administration in dogs undergoing orthopaedic surgery. These regional anaesthesia techniques can therefore reduce the stress response improving the postoperative outcome. When fentanyl is administered in absence of surgical stimulation it does not produce an increased stress response or higher incidence of dysphoria compared to the administration of
Key words: Stress, cortisol, blood glucose, dysphoria, pain, regional
2.1- Stress Response
Stress response is the normal biological adaptive response of an individual, when an external or internal stimulus is perceived to be a threat to its homeostasis. In this response, the stimulus perceived as a threat represents the stressor (Broom and Johnson 1993; Moberg 2000), to which the body responds with a complex systemic reaction which encompasses a wide range of behavioural, endocrinological, immunological and haematological effects. This response originally developed to allow injured animals to survive while they could not have access to food by catabolising their own stored body fuels, but it has been argued that it could be unnecessary or even harmful when associated to surgery (Desborough 2000).
Four main different components can be distinguished in the stress response: the behavioural, the autonomic nervous system, the neuroendocrine and the immune responses (Moberg 2000). These four different components are strictly correlated and coordinated by the hypothalamus and Corticotropin Releasing Hormone (CRH) (Rushen 2000).
The behavioural response is often the best way for the animal to cope promptly with the stressor. For instance removing itself from the threat could represent a good solution, which is derived from the natural predator avoidance behaviour. However, this is not always possible, particularly for domestic animals that often live in a confined environment. Some behaviour that the animal displays during the coping response, such as communicative behaviours, may provide some measure of the internal or subjective state of an animal, and so may be useful as measures of stress (Rushen 2000). If the stressor persists the individual can display behaviours that are out of context and non functional. These displacement behaviours can help the animal to cope with the psychological aspect of the stressor and in turn alleviate some
of the physiological response (Moberg 1985; Moberg 2000; Rushen 2000).
Disease is always associated with changes in behaviour such as disappearance of normal behaviours or appearance of new behaviours. These changes are often considered abnormal behaviours, indicating illness and/or pain. Differentiating between normal and abnormal behaviours can be based on several aspects such as appropriateness of specific behaviours in a given context, appropriateness of the frequency, the severity or the duration of a behaviour in a given context, and the behavioural sequence (normal or altered). The context in which the behaviour occurs allows the clinician to distinguish between appropriate and inappropriate behaviours. Aggression, for example, can be an appropriate response in some contexts (Overall 1997), and serves different purposes depending on context. Fear and anxiety commonly result in stress response. The range of responses to stressors seen in dogs can include avoidance, defensive aggression, panting, salivation, pacing, excessive activity, visual scanning, elimination, dilated pupils, vocalisation, hiding, seeking out human contact, seeking out contact with other dogs or pets, attention-seeking behaviours such as pawing at a person, lowered body posture, flattened ear position, low tail position, anorexia, and digging. Behaviour analysis for stress evaluation in dogs has been routinely used in the last decades. States of movement, body postures, vocalisations, oral behaviours, communicative and exploratory behaviours, or even displacement behaviours (circling, pacing, tail chasing, excessive licking) have been used as indicators of welfare conditions (Hubrecht 1995; Hardie et al 1997; Beerda et al 1997, 1998, 1999; Horvath 2007; Haverbeke 2008). Behaviour evaluation provides an inexpensive and immediate tool for stress assessment, especially useful in those situations where a prompt intervention to control stress is needed, e.g. pain evaluation.
The autonomic nervous system response is promptly activated during an acute stress and it usually has a short duration. It is implicated in the fight or flight response. The magnitude of the autonomic nervous system response can be easily assessed by measuring the increase of serum catecholamines, serum glucose, heart rate and blood pressure. These variations represent good tools to assess the magnitude of an acute response, but they are not accurate for the evaluation of a long-term one. This aspect, make the autonomic nervous system response of less interest in stress assessment (Broom and Johnson 1993; Moberg 2000).
On the contrary, the neuroendocrine response (see Fig. 2.1), derived from the activation of Hypothalamic-Pituitary-Adrenal (HPA) axis, is a long-term response. Therefore, its persistent activation could affect the animal well-being. This neuroendocrine axis holds a great importance in regulating physiologic function like immune competence, reproduction, metabolism and behaviour. Many hormones are involved in the control of the neuroendocrine response.
CRH = Corticotropin Releasing Hormone; DA = Dopamine; GHRH = Growth Hormone –
Releasing Hormone; GnRH = Gonadotropin – Releasing Hormone; SS = Somatostatin; TRH = Thyrotropin – Releasing Hormone; VP = Vasopressin; + = stimulatory hypothalamic factor; - = inhibitory hypothalamic factor; ACTH = Adrenocorticotropic Hormone; FSH = Follicle – Stimulating Hormone; GH = Growth Hormone; LH = Luteinizing Hormone; PRL = Prolactin;
TSH = Thyroid – Stimulating Hormone; IGF = Insulin – like Growth Factor.
Figure 2.1: Hypothalamic – Pituitary neuroendocrine axes and its major biological effects
(adapted from Matteri et al 2000).
Corticotropin Releasing Hormone (CRH) and Vasopressin (VP), produced in the hypothalamus after the perception of a threat, stimulate the pituitary gland to produce Adrenocorticotropic Hormone (ACTH), which acts on the adrenal cortex, stimulating the production of glucocorticoids (cortisol and corticosterone). Among several different actions, these hormones cause an increase in circulating glucose. ACTH, glucocorticoids and glucose have been proved to be good parameters to assess stress (Broom and Johnson, 1993; Matteri et al 2000; Moberg 2000). Other hormones for which secretion is regulated by the HPA axis, like prolactin (PRL) and growth hormone
Cortisol is a biomarker commonly used for stress evaluation in dogs (Beerda et al 1996, 1997, 1998; Coppola et al 2006; Horvath 2007; Haverbeke 2008). It offers the advantage of being a sensitive and universally accepted indicator of stress, easily and inexpensively measurable by commercial kits. Glucose can also be used as a biomarker to assess the HPA axis response to stress. However, because this metabolite is also influenced by the SNA response and other factors, e.g. feeding and starvation, it is not as reliable as cortisol (Matteri et al 2000; Mormede et al 2008). Nevertheless, its use in conjunction with cortisol determination may lend additional support to the assessment of the HPA axis response to stress.
2.1.1-Perioperative stress
Trauma and surgery are potent triggers of stress response in all animals (Desborough 2000). From the first description by Cuthberston in the late 1920s of a generalised metabolic reaction of the body to bone fracture and immobilisation to the characterisation of the role of the HPA axis by Hune in the early 1050s, surgeons and scientists have been dealing with need to improve surgical recovery by understanding the perioperative stress response (Douglas 2002; Butler 2003). Paradoxically, some of the first classical studies about perioperative stress were realised using dogs as experimental animals (Hume 1953; Egdhal 1959). In these studies the adrenal cortisol response to limb injury in dogs was studied. In animals with an intact sciatic nerve or spinal cord, operative injury or superficial burn caused an immediate and sustained increase of adrenal hormones. If the nerve or cord were transacted, the response was abated. In later studies the role of the hypothalamus and the pituitary gland in this response were clarified (Hume 1953).
In humans, extreme hormonal and metabolic responses to stress are associated with increased morbidity and mortality and epidural and spinal anaesthesia are known to modulate the stress response (Douglas 2002). However, data regarding mortality rates are controversial, and recent meta-analysis failed in detecting a connection among regional anaesthesia and mortality, although a reduction in the morbidity was confirmed (Kettner 2011; Chen 2013). Specifically, perioperative stress response has adverse effects on immune function, which could increase the postoperative susceptibility to infections and the chances of prolonged ileus; moreover it can predispose to hypercoagulability status, increase the risk of ischemia and reperfusion injury, as well the systemic inflammatory responses (Liu et al 1995; Wolf 2012). There is also a reduction in deep vein thrombosis, pulmonary embolism, blood loss, pneumonia, respiratory depression, myocardial infarction and renal failure (Rogers et al 2000). In another study by Rasmussen et al (2005) it was demonstrated that the alteration of the neuroendocrine system related to the perioperative stress response could be a contributing factor in the development of postoperative cognitive dysfunction in elderly people.
Using neuraxial blockade and β-adrenergic blockade, preventing hypothermia and using appropriate opioid treatment for analgesia have been demonstrated to be effective tools to reduce postoperative stress and to favour a safe and prompt recovery (Douglas 2002). These elements are combined with a minimally invasive operative technique and aggressive postoperative rehabilitation (e.g. enteral nutrition and ambulation) in so called “Fast-track surgery” in humans. This method of care has been shown to reduce the stress response and associated organ dysfunction; it optimises recovery and prompts early hospital discharges (Brodner et al 2001; Kehlet and Wilmore 2002).
In the last decades, surgery and related procedures have also been recognised as major stressors in veterinary medicine (Hansen et al
procedures represent a major source of stress for the animal, due to the surgery itself and various associated elements, such as pain, analgesia and anaesthesia-induced dysphoria, human handling and confinement in a hospitalisation unit (Hetts et al 1992; Hansen et al 1997; Hardie et al 1997; Mellor et al 2000; Wells 2004).
Surgical stress and that associated with related procedures has been evaluated in dogs using different markers. Cortisol and behavioural analysis have often been used (Hetts et al 1992; Beerda et al 1997; Hansen et al 1997; Hardie et al 1997; Beerda et al 1998; Vaisanen 2002). Pain is a potent stressor (Desborough 2000). Four different types of scoring system for acute pain are currently used for postoperative behavioural evaluation: the Visual Analogue Scale (VAS), the Simple Descriptive Scale (SDS) and the Numerical Rating Scale (NRS) and composite scales. A SDS has four or five degrees of severity (ex, No evidence of pain, Mild, Moderate, Severe, Very Severe) (Firth and Aldane 1999; Hellyer 2005). The SDS is easy to use but it does not allow small changes in pain response to be assessed. The NRS may be produced by assigning a numeric score to each of the categories of SDS. A NRS may include descriptive definitions of each category of pain (Hardie et al 1997) but it often provides no real improvement in accuracy over a SDS; the numeric score simply facilitates tabulation or analysis of results (Firth and Aldane 1999). The VAS is a simple scale, consisting of a straight line (usually 100 mm, horizontal or vertical) on paper, with a description of the limits of the scale written at each end (ex, No pain, Severe Pain). The observer places a mark somewhere along the line to interpret the degree of pain. The VAS is subject to a great degree of observer variation, but because it does not use defined categories, it is often considered to be more sensitive than a NRS or SDS (Lascelles et al 1998; Firth and Aldane 1999). Composite scales have been developed combining some aspects of the different scales (Holton et al 2001; Firth and Aldane 1999). They use specific validated behavioural categories and physiological parameters to assess pain. A
numerical score can be assigned, but there are no validated criteria to assign a score to a specific category for many of these scales (Holton et al 2001; Firth and Aldane 1999; Hellyer 2005).
An exception among composite scales is represented by the Glasgow Pain Scale (Figure 2.2). It is a behaviour-based questionnaire developed and validated to measure acute pain in dogs (Reid et al. 2007). The GPS uses well-defined behavioural categories to describe the behaviour of dogs in pain and assigns a scientific-based specific weight to each category for pain scoring (Holton et al 2001; Morton et al 2005).
Figure 2.2: Short Form of the Glasgow Composite Pain Scale (Reid et al. 2007). SHORT FORM OF THE GLASGOW COMPOSITE PAIN SCALE
Dog’s name _______________________________ Hospital Number __________ Date / / Time Surgery Yes/No (delete as appropriate)
Procedure or Condition_____________________________________________
______________________________________________________________
In the sections below please circle the appropriate score in each list and sum these to give the total score.
A. Look at dog in Kennel
Is the dog?
D. Overall
Is the dog?
(v)
Happy and content or happy and bouncy 0
Quiet 1
Indifferent or non-responsive to surroundings 2 Nervous or anxious or fearful 3 Depressed or non-responsive to stimulation 4
(ii)
Ignoring any wound or painful area 0 Looking at wound or painful area 1 ing wound or painful area 2 Rubbing wound or painful area 3 Chewing wound or painful area 4 Lick Groaning 2 (i) Quiet 0 Crying or whimpering 1 Screaming 3
In the case of spinal, pelvic or multiple limb fractures, or where assistance is required to aid locomotion do not carry out section B and proceed to C
Please tick if this is the case then proceed to C.
C. If it has a wound or painful area
including abdomen, apply gentle pressure 2 inches round the site.
Does it?
(iv)
Do nothing 0 Look round 1 Flinch 2 Growl or guard area 3
Snap 4
Cry 5
B. Put lead on dog and lead out of the kennel.
When the dog rises/walks is it?
(iii) Normal 0 Lame 1 Slow or reluctant 2 Stiff 3 It refuses to move 4 Is the dog? (vi) Comfortable 0 Unsettled 1 Restless 2 Hunched or tense 3 Rigid 4
2.1.2-Stress and Anaesthesia
Anaesthetic management can be crucial on stress response. In fact, specific protocols have been shown to control stress better than others. Specifically, a large number of studies show that neuraxial anaesthesia can prevent the endocrine and metabolic response to surgery (Wolf et al. 1993; Liu et al. 1995; Meissner et al. 1997; Sibanda 2006). Spinal anaesthesia provides a more profound degree of blockade compared to epidural anaesthesia by blocking both afferent impulse from the surgical site to the central nervous system and efferent autonomic pathways to the liver and the adrenal medulla, thus completely abolishing the adrenocortical and glycaemic responses to surgery (Wolf et al. 1998; Wolf 2012). In veterinary medicine, epidural anaesthesia can lower the neuroendocrine stress response in dogs undergoing femoro-tibial joint surgery when compared to systemic administration of opioids (Sibanda 2006).
In humans, opioids have been shown to control the stress response associated with surgery only at very high doses (fentanyl 50-100 µg/kg; morphine 4 mg/kg) (Liu et al. 1995; Desborough 2000). Considering that commonly used doses of these drugs are 5-10 µg/kg for fentanyl and 4 mg/kg for morphine, it appears clear that doses 10 times higher than therapeutic doses are extremely likely to be associated to severe side effects.
The administration of medetomidine has been associated with a better control of the stress response in dogs when compared to acepromazine (Väisänen et al. 2002).
No studies have been performed in dogs or in humans to assess the role of peripheral nerve blocks on perioperative stress.
2.1- Consequences of stress
2.2.1-Cardiovascular system
The activation of the sympathetic nervous system may result in myocardial ischaemia and infarction (Rocco et al 1987; Mulcahy et al 1988; Willich et al 1989). Similarly, interventions that inhibit the sympathetic response can reduce cardiac morbidity (Tofler et al. 1984; Brezinski et al 1988; Fujita and Franklin 1987). Because sympathetic activation occurs during the perioperative period, inhibition of sympathetic activity may reduce postoperative myocardial morbidity. The mechanisms whereby sympathetic activation causes cardiac morbidity can be mediated both by increases in myocardial oxygen demand or reductions in myocardial oxygen supply. For example, pain from surgical stimuli can activate sympathetic efferent nerves and increase heart rate, inotropy, and blood pressure. Although activation of the sympathetic nervous system can increase myocardial oxygen demand and result in ischaemia, most episodes of myocardial ischaemia occur in absence of major haemodynamic changes. (Slopoff and Keats 1989: Hollenberg et al 1992; Leung et al 1990) In fact, most episodes of ischaemia are not proceeded by increases in myocardial oxygen demand, with the exception of small increases in the heart rate (Chierchia et al 1980; Chierchia et al 1983). Thus, reductions in myocardial oxygen supply may be the primary cause of silent ischaemia. Reduction in myocardial oxygen supply may result from coronary vasoconstriction or thrombosis in coronary arteries and may be exacerbated by episodes of perioperative hypoxemia (Rosembeg et al 1990; Reeder et al 1991). Activation of the sympathetic nervous system may trigger both of these mechanisms, with resultant signs of
myocardial ischaemia such as ST-segment changes, arrhythmias, and myocardial infarction (Blomberg et al 1989; Schwartz 1980; Flatley et al 1985). Sympathetic activation has also been proposed to cause postoperative hyper-coagulable states (Rosenfeld 1993) and thus may be a factor in thrombotic causes of myocardial ischaemia.
2.2.2-Coagulation
Major surgery is associated with a hypercoagulable state that persists well into the postoperative period (Ygge 1970; Collins et al 1977; Donadoni et al 1989). Increases in perioperative coagulation are associated with vaso-occlusive and thromboembolic events that may result in postoperative morbidity and mortality (Tuman et al 1991; Christopherson et al 1993). Although the aetiology of his postoperative increase in coagulability is uncertain, the stress response appears to be an important initiator (Rosenfeld 1993; Breslow 1993). Post-operative changes occur in all arms of the coagulation system and include increased concentration of coagulation factors (Collins et al 1977), increased concentration of coagulation inhibitors (Anderson et al 1987), enhanced platelet activity (O’Brien et al 1974) and impaired fibrinolysis (Ygge 1970; Anderson et al 1987).
2.2.3-Pulmonary function
Post-operative pulmonary dysfunction occurs as a result of surgery and anaesthesia-related physiologic perturbations and is a major cause of postoperative morbidity. Upper abdominal and thoracic incisions
1973; Ali et al 1974; Craig 1981), while laparoscopic and peripheral operations have little effect (Rademaker et al 1992; Couture et al 1994). Pulmonary dysfunction after upper abdominal surgery occurs because of pain (Craig 1981), abnormal diaphragmatic function (Pansard et al 1993), and increased abdominal and lower intercostal muscle tone during exhalation (Duggan and Drummond 1987). Pulmonary dysfunction begins with incision and remains diminished for 7-14 days postoperatively (Meyers et al 1975). The most important alteration of respiratory function is decreased functional residual capacity, which begins about 16 hours postoperatively, reaches a peak at 24-48 hr, and usually resolves within 1 week (Ali et al 1974). Decreased functional residual capacity may result in atelectasis and ventilation-perfusion abnormalities leading to hypoxemia, pneumonia, and post-operative pulmonary complications (Craig 1981). Patients especially at risk for reduction of functional residual capacity and resultant pulmonary complications are those with pre-existing pulmonary disease (Tarhan et al 1973), upper abdominal and thoracic incisions (Rademaker et al 1992), advanced age (Wahba 1983), obesity (Rawal et al 1984) and those in severe pain. Choice of anaesthetic technique affects the degree of post-operative pulmonary dysfunction. Use of general anaesthesia may briefly exacerbate surgery induced pulmonary dysfunction (Sydow 1988). Mechanical ventilation, paralysis, inhaled anaesthetic and opioids all contribute to reduce pulmonary function (Nunn 1990). Previous studies suggest that the use of epidural anaesthesia has the potential to reduce pulmonary morbidity by providing better analgesia, improved diaphragmatic function and reduced frequency and severity of postoperative hypoxemia (Rademaker et al 1992; Cuschieri et al 1985).
2.2.4-Gastrointestinal system
Postoperative ileus is a temporary impairment of gastrointestinal motility that occurs after surgery. Although most common and severe after major abdominal procedures, ileus also occurs after peripheral operations, general trauma or other stressful situations (Livingston and Passaro 1990). Ileus delays resumption of an enteral diet, and this delay may contribute to post-operative morbidity. An early feeding has been shown to reduce the surgical stress response, reduce postoperative septic complications and improve wound healing (Moore et al 1992; Shou et al 1994). Anaesthetic or analgesic techniques that speed recovery or prevent postoperative ileus have the potential to substantially reduce post-operative morbidity. Post-operative ileus affects all segments of the gastrointestinal tract with variable duration and intensity (Wells et al 1961). Although the pathophysiologic features of postoperative ileus are not fully understood, the most commonly accepted theory is that pain activates a spinal reflex arc that inhibits the intestinal motility (Furness and Costa 1974). In addiction, surgical stress induces sympathetic hyperactivity and excessive sympathetic stimulation of the bowel inhibits organised propulsive activity (Livingston 1990). Regional anaesthesia can reduce postoperative ileus through relief of pain (Wattwil 1988), systemic absorption of local anaesthetic (Rimbach 1990) and reduction of requirements for systemic opioids (Schang 1986).
2.2.5-Immune function
immunosuppression is unclear, many known mediators of the stress response are potent immunosuppressant (Davis et al 1991). Post-operative immunosuppression typically lasts for several days, may last longer in inherently immunosuppressed patients (Scannel 1983; Lennard et al 1985), and may predispose to the development of postoperative infections (Meakins 1991; Moss et al 1988), or facilitate postoperative tumour growth and metastases (Lundy et al 1978; Tanemura et al 1982; Eggermont et al 1987). General anaesthetics with the exception of large doses of opioids, cannot suppress the stress response and may exacerbate post-operative immunosuppression by depression of cellular and humoral immune function (Kehlet et al 1977; Markovic et al 1993; Stevenson et al 1990). For example, immunosuppression resulting from general anaesthetics occurs within 15 minutes after induction and may persist for as long as 3-11 days (Tonnesen and Wahlgreen 1984; Markovic et al 1993). Spinal and epidural anaesthesia reduce the peri-operative stress response improving immune function (Wolf et al. 1998; Wolf 2012).
2.3- Fentanyl
Fentanyl is a short-acting μ agonist opioid that is approximately 100 times more potent than morphine. Due to its short half-life it is generally used in constant or variable infusion regimens, and it is very commonly used to provide peri-operative analgesia in dogs (Pascoe 2000; Lamont & Mathews 2007). It has an isoflurane-sparing effect in dogs undergoing surgery, however it is associated with several side effects which include bradycardia, hypotension, hypoventilation, ileus, dysphoria, nausea and vomit (Lamont & Mathews 2007; Becker et al. 2012; Keating et al. 2013).
2.4- Spinal anaesthesia
Spinal (intrathecal) anaesthesia consists in the administration of local anaesthetics and/or opioids into the subarachnoid space (Stanley 1915). It is a reliable technique to provide effective regional anaesthesia and analgesia during hind limb surgery in dogs (Otero & Campoy 2012; Sarotti et al. 2012). It produces a more profound degree of blockade and has some significant advantages compared to epidural anaesthesia: faster onset and offset of action, and lower systemic absorption of drugs. Several variables affect cranial spread of LA after subarachnoid administration including dose, volume and baricity of the injected solution, as well as injection site, direction of the needle, cerebrospinal fluid density and the position of the patient (Greene 1985). Local anaesthetics can be administered in the subarachnoid space to achieve a sensory and motor block. In humans, spinal administration of lidocaine recently has fallen into disuse, because of its potential neurotoxicity (Freedman et al. 1998; Hogson et al. 1999). Opioids such as morphine or fentanyl can also be used as adjuvants to improve quality and increase duration of the block, to better control the cardiovascular response to surgery (Samii et al. 1981; Ben-David et al. 1997) and to achieve longer lasting postoperative analgesia.
The potential side effect associated with the spinal administration of local anaesthetics can include nerve damage and hypotension due to sympathetic blockade, while urinary retention, pruritus, respiratory depression and nausea can be associated with the intrathecal administration of opioids as adjuvant analgesics (Sarotti et al. 2011; Otero & Campoy 2012).
2.5- Peripheral Nerve Bocks
Peripheral regional anaesthesia consists in the administration of a local anaesthetic, sometimes in combination with other drugs, in correspondence to a peripheral nerve. These techniques have been used in the last years to provide analgesia to specific areas in different clinical situations in dogs (Campoy et al. 2010; Campoy et al. 2012(1); Vettorato et al. 2012; Portela et al. 2013(1); Portela et al. 2013(2); Portela and Romano 2014). It has been shown that peripheral nerve blocks can provide the same degree of analgesia compared to neuraxial blocks, but with lower incidence of side effects (Davies et al. 2004; Campoy et al 2012(2); Caniglia et al 2012). Specifically, the combination of femoral and sciatic nerve blocks have been demonstrated to be an effective alternative to neuraxial anaesthesia during hind limb surgery in dogs (Campoy et al. 2012 (1); Vettorato et al. 2012; Portela et al. 2013). Recent approaches are assisted by the use of nerve stimulator and/or ultra-sound machines, used to identify specific nerves, which can help reducing the incidence of nerve damage while performing these techniques (Campoy et al. 2010; Campoy et al. 2012; Portela et al. 2013(1); Portela et al. 2013 (2); Portela and Romano 2014).
2.6- Aims of the study
The objectives of the present study were:1. To evaluate the role of different ways of administration of premedication drugs on the stress response in dogs.
2. To assess stress response, dysphoria, and pain in dogs undergoing orthopaedic stifle surgery, treated with different analgesic protocols. 3. To establish if fentanyl administration is related with increased stress response and dysphoria.
2.7- Hypotheses
The hypotheses of the present study were:1. That intramuscular administration of premedication drugs may decrease the stress response in comparison with intravenous administration of the same drugs.
2. That spinal anaesthesia might completely control the stress response associated with orthopaedic surgery in dogs, following similar mechanisms to those described in humans.
3. That Peripheral nerve block could reduce the stress response compared to fentanyl administration in dogs undergoing orthopaedic surgery.
4. That both spinal and peripheral nerve blocks might reduce the incidence of post-operative dysphoria compared to fentanyl administration.
5. That regional anaesthesia techniques, providing better analgesia, could reduce the pain scores and therefore decrease the opioid consumption after orthopaedic surgery in dogs.
6. That fentanyl might determine a high incidence of dysphoria, and therefore a stress response when administered in dogs not undergoing surgery.
3-Stress and anaesthetic
management
3.1- Materials and Methods
3.1.1-Animals
The present prospective, blind clinical study was conducted in accordance with the national legislation on protection of animals used for scientific purposes (D.Lgs.vo 116/92) and was approved by the Institutional Animal Care and Use Committee of the University of Pisa (Prot. #1397).
Seventeen healthy dogs undergoing non invasive orthopaedic diagnostic procedures (computed tomography or X-ray) under general anaesthesia at the Veterinary Teaching Hospital Mario Modenato (University of Pisa) were included in this study after obtaining owner’s written consent. Physical, haematological and biochemical evaluation were used to assess the good health status, and thus dogs classified as having a physical status 1 or 2, according to the American Society of Anaesthesiologists (ASA), were enrolled in the study. All animals were Labrador Retrievers or Golden Retrievers belonging to the Guide Dog for the Blind school. Animals showing a pre-anaesthetic behaviour score of 4 (Table 3.1), receiving concurrent medications, or ASA physical status ≥ 3 were excluded from the study.
The dogs included in the study were randomly divided into two groups based on the route of administration of the pre-medication drugs through a drawing lot as follows:
• IM group (n=8): Pre-medication was administered intramuscularly
• IV group (n=9): Pre-medication was administered intravenously Using a descriptive four point scale, previously described by Becker et al. (2012), a pre-anaesthesia score behaviour was assigned to each dog at the arrival at the Clinic (Table 3.1).
3.1.2-Anesthetic management
Food, but not water was withheld to all animals 12 hours prior surgery. All dogs received 0.1 mg kg-1 of methadone (Eptadone. Molteni & Flli
Alitti S.p.a., Italy) and 10 µg kg-1 of acepromazine (Prequillan; Fatro
Spa, Italy) as preanesthetic medication. Dogs in IM group received the premedication intramuscularly as soon as they were presented to the anaesthesia service. Twenty minutes after administration of the premedication, a catheter was placed in the cephalic vein and general anaesthesia was induced with 4-6 mg kg-1 of propofol IV (Propofol
Kabi; Frenesius Kabi Italia Srl Italy) and maintained with isoflurane (Isoflo, Esteve Spa, Italy) in oxygen delivered through a semi-closed breathing circuit system after tracheal intubation. The dogs belonging to the IV group were positioned in a kennel for 20 minutes upon presentation at the anaesthesia service. After that time, an intravenous cephalic catheter was placed and premedication was administered intravenously. Five minutes later, general anaesthesia was induced with 4-6 mg kg-1 of propofol IV (Propofol Kabi; Frenesius Kabi Italia Srl
Italy) and maintained with isoflurane (Isoflo, Esteve Spa, Italy) in oxygen delivered through a semi-closed breathing circuit system after tracheal intubation. Lactate Ringer’s solution was infused at a rate of
3-5 mL kg-1 hr-1 throughout the anaesthetic period. Continuous ECG,
heart rate (HR), respiratory rate (fR); pulse oximetry, indirect blood pressure (iBP), end-tidal CO2 (PE’CO2) and end-tidal isoflurane concentration (FE’ISO) were continuously monitored throughout anaesthesia every 5 minutes using a multi-parametric monitor. Five minutes after induction of general anaesthesia, all dogs were positioned for the diagnostic procedure.
Dogs were continuously monitored to detect spontaneous movements or any change in the cardiovascular parameters in response to stimulations. If spontaneous movements were detected, dogs received 0.5 to 1 mg kg-1 of propofol IV. If HR or dMAP increased more than
20% compared with Tb an IV bolus of 2.0 µg kg-1 of fentanyl was
administered as rescue analgesia.
3.1.3-Post-operative evaluation
After tracheal extubation, dogs were positioned in a kennel. The postoperative evaluation was performed when the dogs became aware of the surrounding by a researcher unaware of the group the dog belonged to. Recovery quality was scored in all groups based on behaviours displayed shortly after extubation, using a descriptive four point scale (Table 3.1) previously described by Becker et al. (2012).
3.1.4-Sample collection
In order to measure serum cortisol and blood glucose level, blood samples were collected in all dogs at arrival in the Hospital (TAr), at
induction of general anaesthesia, after endotracheal intubation (TINT),
immediately after tracheal extubation (TEXT), and one hour after
tracheal extubation (TPST).
Blood glucose concentration was measured immediately after sample collection with a glucometer (Abbot Freestyle Optium, Abbott Laboratories, Illinois-USA) and the results were reported in milligrams per deciliter (mg dL-1). A 5 mL aliquot of blood was dispensed into a serum separator tube. Immediately after clot formation occurred, the sample was centrifuged at 3000 rpm for 10 minutes, and serum separated and stored at -80°C in labeled Eppendorf tubes. Cortisol concentration was measured by a solid phase radioimmunoessay (ST Aia Pack, Tosoh Bioscience, Japan).
The personnel measuring the serum cortisol and blood glucose level were unaware of the group assigned to each sample.
3.1.5-Statistical analysis
Data were evaluated for normal distribution by the Shapiro-Wilk normality test. Age weight dysphoria and preanesthetic scores were compared using a Mann Whitney test. A Kruskal-Wallis test and a Dunns post hoc test for multiple comparisons was used to compare cortisol levels between IM and IV groups. Differences were considered statistically significant with P < 0.05. Data were expressed as median (min-max).
Behavior scoring criteria
Behavior scoring criteria
Pre-anesthesia Scores
Pre-anesthesia Scores
1 Calm dog/may be alert and attentive (no panting/pacing or barking)
2 Mildly excited when someone is present/controlled barking/pacing/
pulling
3 Excited or exuberant: jumps up/barking/pacing/panting/ lip licking, but
seems to calm down with attention
4 Whining/does not want to be in kennel/aggressive/personnel are unable
to interact with dog to calm it
Recovery Scores
Recovery Scores
1 Dog is quiet or raises head calmly, does not appear agitated
2 Dog transiently pants or whines and/or appears to gently paddle with
front feet immediately upon extubation, but then settles
3 Dog is panting/whining or whimpering/occasionally
4 Dog is agitated, trying to bite or thrashing body in an uncoordinated
manner, does not seem to be “present”
3.2- Results
No significant differences were evinced among groups for weight (p=0,19), age (p=0,79), preoperative behaviour score (p=0,84) or recovery median scores (p=0,99) (Table 3.2).
The trends of the blood glucose level during the different time points and the comparison among groups are represented in the Fig. 3.1, and there was no significant increase at any time point in any group.
Cortisol levels evaluated during the study are represented in the Fig. 3.2. Cortisol basal levels (TAr) were within the normal range reported
for dogs (Nelson 2006) in all dogs, and did not increase significantly at any time point in any case.
IM IV
Weight (kg) 30 (28-35) 30 (25-35)
Age (months) 15 (12-18) 18 (9-19)
Pre-operative behaviour score 1 (1-2) 1 (1-3)
Recovery score 1 (1-3) 1 (1-2)
Table 3.2: Results of weight, age, pre-anaesthetic behaviour score and recovery score in the two
Figure 3.1: blood glucose levels (mg/dl) at different time-points (T0=arrival at the Hospital;
Figure 3.2: Serum cortisol (µg/dl) at different time-points (T0=arrival at the Hospital; T1=tracheal
3.3- Discussion
The results obtained in the present study show that glucose and cortisol levels do not change in association with the different premedication administration routes used, remaining within normal limits described in dogs at all time points. No differences were evinced in the recovery scores among the two groups. These results suggest that the intramuscular administration of the selected drugs does not determine a reduction in the stress related biomarkers, nor it improves the quality of the recovery when compared to intravenous administration, therefore there is no clear indication for one administration route or the other.
4-Stress, dysphoria and pain
in dogs undergoing stifle
4.1- Materials and Methods
4.1.1-Animals
The present prospective, blind clinical study was conducted in accordance with the national legislation on protection of animals used for scientific purposes (D.Lgs.vo 116/92) and was approved by the Institutional Animal Care and Use Committee of the University of Pisa (Prot. #1397).
Forty-five dogs submitted to tibial tuberosity advancement (TTA) or tibial plateau levelling osteotomy (TPLO) and 15 healthy dogs undergoing non invasive orthopaedic diagnostic procedures (computed tomography or X-ray) under general anaesthesia at the Clinica Veterinaria Apuana were included in this study after obtaining owner’s written consent. Physical, haematological and biochemical evaluation were used to assess the good health status, and thus dogs classified as having a physical status 1 or 2, according to the American Society of Anaesthesiologists (ASA), were enrolled in the study. Animals weighting less than 6 or more than 40 kg, aged less than 1 or more than 10 years, showing a pre-anaesthetic behaviour score of 4 (Table 4.1), receiving concurrent medications, with clotting, neuromuscular or neurologic disorders, skin infections, or ASA physical status ≥ 3 were excluded from the study.
Fifteen healthy dogs with no sign of pain or lameness undergoing hip and elbow dysplasia diagnostic procedures were included as control group (CTR Group), while 45 dogs undergoing surgery were randomly divided into three groups of 15 dogs based on the analgesic protocol through a drawing lots as follows:
• PNB Group (n=15): received a peripheral nerve block of the femoral and sciatic nerve
• FEN Group (n=15): received a fentanyl variable rate infusion. Using a descriptive four point scale, previously described by Becker et al. (2012), a pre-anaesthesia score behaviour was assigned to each dog at the arrival at the Clinic (Table 4.1).
4.1.2-Anesthetic management
Food, but not water was withheld to all animals 12 hours prior surgery. An intravenous catheter was aseptically placed in a cephalic vein, and all dogs received 0.1 mg kg-1 of methadone IV (Eptadone. Molteni &
Flli Alitti S.p.a., Italy) and 4 mg kg-1 of carprofen SQ (Rimadyl; Pfizer
Italia Srl Italy) as preanesthetic medication. Five minutes after methadone administration, general anaesthesia was induced with 4-6 mg kg-1 of propofol IV (Propofol Kabi; Frenesius Kabi Italia Srl Italy)
and maintained with isoflurane (Isoflo, Esteve Spa, Italy) in oxygen delivered through a semi-closed breathing circuit system after tracheal intubation. Lactate Ringer’s solution was infused at a rate of 3-5 mL kg-1 hr-1 throughout the anaesthetic period. An intra-arterial catheter
was placed in the dorsal pedal artery (Picture 4.1) to measure direct arterial pressure (dBP). Continuous ECG, heart rate (HR), respiratory rate (fR); pulse oximetry, direct blood pressure (dBP), end-tidal CO2 (PE’CO2) and end-tidal isoflurane concentration (FE’ISO) were continuously monitored throughout anaesthesia every 5 minutes using a multi-parametric monitor (Criticare Poet Plus 8100, Criticare Systems, USA) (Picture 4.2). Body temperature was maintained above 37°C using electrically heated and thermal foil blankets (Picture 4.3 and 4.4). For dBP measurements, the transducer was zeroed at the level of the right atrium. Volume controlled ventilation was provided to maintain PE’CO2 between 35 and 45 mmHg (Picture 4.5).
4.1.3-Analgesic protocols
After induction of general anaesthesia, the hair of the entire affected pelvic limb, the lumbar and gluteal region was clipped in all dogs submitted to surgery.
Dogs receiving peripheral nerve blocks (PNB Group) were positioned in lateral recumbency with the affected limb uppermost. The dorso-lateral lumbar and sacro-gluteal region were aseptically prepared and the femoral and sciatic nerve were blocked with a lateral pre-iliac and a parasacral approach respectively, as described elsewhere (Portela et al. 2010; Portela et al. 2013) (Picture 4.6A and 4.6B). Briefly, the femoral nerve was approached inserting an insulated needle (Stimuplex, BBraun Germany) connected to a nerve stimulator (Stimuplex HNS12, BBraun Germany) (Picture 4.7) from the lateral aspect of the lumbar muscles and cranially to the iliac crest. The injection point was located at the intersection of two imaginary lines: the first drawn from the spinous process of the 6th lumbar vertebra, perpendicular to the spine in a dorso-ventral direction and the second parallel to the spine starting from the most cranial aspect of the iliac crest until it crossed the first line. The nerve stimulator was initially set to 1.5 mA (2Hz, 0.1 ms) and the needle advanced in a caudo-medial direction with a 30-45° angle until contractions of the quadricep muscle were evoked (extension of the stifle joint) due to femoral nerve stimulation. The stimulating current was then gradually reduced and when necessary the needle repositioned until proper muscular response was obtained with 0.3 to 0.5 mA, and thus 0.15 mL kg-1 of 0.5% bupivacaine
(Bupisolver, Piramal Critical Care It, Spa, Italy) was injected. Subsequently, to block the sciatic nerve an imaginary line was traced between the dorsal part of the iliac crest and the ischiatic tuberosity. This line was divided in three equal segments and the injection point
The stimulating needle (1.5 mA, 2Hz, 0.1 ms) was introduced in this site, perpendicular to the skin plane deep enough until the contractions of the gastrocnemius muscle or even digital (and/or tarsus) flexion or extension were evoked due to the sciatic nerve stimulation. The stimulating current was gradually reduced and when necessary the needle repositioned until the proper muscular response was obtained with 0.3 to 0.5 mA, and thus 0.15 mL kg -1 of 0.5% bupivacaine were
injected.
Dogs assigned to receive spinal (intrathecal) anaesthesia (SPI Group) were positioned in lateral recumbency with the pelvic limbs pulled cranially. The dorsal lumbosacral region was then aseptically prepared. Spinal anaesthesia was performed using a paramedian approach at the L5-L6 intervertebral space, using a 22G, 90 mm spinal
needle (Terumo Spinal Needle, Belgium). The spinal needle was introduced laterally to the L6 spinal process until the tip of the needle
reached the dorsal lamina of the 6th lumbar vertebra. Thereafter the
needle tip was walked cranio-medially to reach ligamentum flavum, at this level the stylet was removed and the needle further advanced towards the dorsal subaracnoid space (Picture 4.8). Once the correct position of the needle was confirmed by the flow of cerebro-spinal
fluid in the hub of the needle, 0.05 mL kg -1 0.5% isobaric bupivacaine
was injected.
After instrumentation, dogs were positioned in the surgical position, general anaesthesia was stabilised at FE’ISO 1.2 vol% for 10 minutes
and direct mean arterial pressure (dMAP), HR and FE’ISO were
registered as baseline (Tb). Surgery started 15 minutes after Tb (T0). The
following time points were also marked: five minutes after beginning of surgery (T5); half time of the duration of surgery (T1/2) and end of
surgery (TEND).
Once the procedure began, FE’ISO was gradually reduced by 0.1-0.2
vol% every 10 minutes, until the minimal concentration preventing movement was reached.
Dogs in the fentanyl group (FEN Group) received a loading dose of 5 µg kg-1 (Fentanest, Pfizer Italia S.r.l., Italy) 10 minutes before beginning
the surgery followed by a 10 µg kg-1 h-1 infusion. Intra-operatively, the
dose was adjusted to maintain the HR and dMAP within 20% of the values registered before the beginning of the surgery (Tb). When the
suture of the surgical wound was started, the fentanyl infusion was reduced to 3 µg kg-1 hr-1 and 0.2 mg kg-1 of methadone was
administered IM. Fentanyl administration was interrupted at the end of the surgery.
Dogs undergoing diagnostic procedures and assigned as control group (CTR Group) were positioned for X-ray or CT exams after premedication and induction of general anaesthesia. No medication other than those described in the anaesthetic protocol was administered to these animals. After positioning, anaesthesia was
stabilised at FE’ISO 1.2 vol% for 10 minutes and dMAP and HR were
registered as baseline (Tb). The procedure started 15 minutes after Tb.
4.1.4-Intra-operative evaluation
Dogs were continuously monitored to detect spontaneous movements or any change in the cardiovascular parameters in response to surgical stimulations. If spontaneous movements were detected, dogs received 0.5 to 1 mg kg-1 of propofol IV and the FE’ISO was increased to the
previous level at which movements were not present and maintained at that level until the end of the procedure. If HR or dMAP increased
more than 20% compared with Tb an IV bolus of 2.0 µg kg-1 of fentanyl
was administered as rescue analgesia. If the parameters did not return to the pre-stimulation values or increased again after 10-15 minutes, another bolus of 2.0 µg kg-1 of fentanyl was administered, followed by a
continuos infusion in SPI or PNB groups, or adjustment of the fentanyl infusion rate in the FEN group.
4.1.5-Post-operative evaluation
After tracheal extubation, dogs were positioned in a cushioned kennel. The postoperative evaluation started when the dogs became aware of the surrounding and was performed every 15 minutes for 60 minutes by a researcher unaware of the analgesic technique used during surgery. In the CTR Group, the postoperative evaluation could not be blinded as dog belonging to this group had not undergone surgery, therefore their pelvic limbs were not clipped.
Recovery quality was scored in all groups based on behaviours displayed shortly after extubation, using a descriptive four point scale (Table 4.1) previously described by Becker et al. (2012). Post-operative pain was assessed in all animals undergone surgery using the short-form Glasgow Composite Measure Pain Scale (Reid et al. 2007).
Methadone, 0.1 or 0.15 mg kg-1 IV was administered as rescue analgesia
if pain scores were greater than 5/20 or 10/20, respectively.
4.1.6-Sample collection
In order to measure serum cortisol and blood glucose level, blood samples were collected in all dogs at arrival in the Hospital (TAr), at
induction of general anaesthesia, after endotracheal intubation (TINT),
immediately after tracheal extubation (TEXT), and one hour after
tracheal extubation (TPST).
Blood glucose concentration was measured immediately after sample collection with a glucometer (Abbot Freestyle Optium, Abbott
Laboratories, Illinois-USA) and the results were reported in milligrams per deciliter (mg dL-1). A 5 mL aliquot of blood was dispensed into a serum separator tube. Immediately after clot formation occurred, the sample was centrifuged at 3000 rpm for 10 minutes, and serum separated and stored at -80°C in labeled Eppendorf tubes. Cortisol concentration was measured by a solid phase radioimmunoessay (ST Aia Pack, Tosoh Bioscience, Japan).
The personnel measuring the serum cortisol and blood glucose level were unaware of the group assigned to each sample.
Dogs in PNB and SPI group were evaluated during a follow-up period of 30 days to rule out neurological deficits.
4.1.7-Statistical analysis
Data were evaluated for normal distribution by the Shapiro-Wilk normality test. Distribution of sex and type of surgery among groups were analysed using a Chi square test. Age, weight, cortisol levels, blood glucose level, HR and dMAP were compared among groups using one-way ANOVA and Bonferroni’s post hoc test; while the different time points within each group were analysed by an ANOVA test for repeated measures and a Bonferroni’s post hoc test. End tidal isofluorane concentrations (FE’ISO), pre-operative behaviour, post-operative dysphoria and post-post-operative pain scores were compared using Kruskal-Wallis test and a Dunns post hoc test for multiple comparisons. Methadone doses administered during the post-operative period were compared using a Mann-Whitney test. Differences were considered statistically significant with P < 0.05. Parametric data were expressed as mean ± SD, while non-parametric data as median (min-max).
Behavior scoring criteria
Behavior scoring criteria
Pre-anesthesia Scores
Pre-anesthesia Scores
1 Calm dog/may be alert and attentive (no panting/pacing or barking)
2 Mildly excited when someone is present/controlled barking/pacing/
pulling
3 Excited or exuberant: jumps up/barking/pacing/panting/ lip licking, but
seems to calm down with attention
4 Whining/does not want to be in kennel/aggressive/personnel are unable
to interact with dog to calm it
Recovery Scores
Recovery Scores
1 Dog is quiet or raises head calmly, does not appear agitated
2 Dog transiently pants or whines and/or appears to gently paddle with
front feet immediately upon extubation, but then settles
3 Dog is panting/whining or whimpering/occasionally
4 Dog is agitated, trying to bite or thrashing body in an uncoordinated
manner, does not seem to be “present”
Picture 4.1: Arterial catheter placement in the dorsal pedal artery.
Picture 4.6A: Femoral nerve block with a lateral pre-iliac approach Picture 4.6B: Sciatic nerve block with a parasacral approach
Picture 4.7: Nerve stimulator Stimulplex Bbraun
4.2- Results
No significant differences were evinced among groups for weight (p=0.92), sex (p=0.49), type of surgery (p=0.34) and preoperative behaviour score (p=0.91) (Table 4.2). Dogs of the CTR group were significantly younger that dogs of the other groups (p<0.0001). Duration of the procedure was significantly shorter in the CTR group (p<0.0001) compared to the other groups. Surgical duration did not differ significantly among PNB, SPI and FEN groups (p=0.45) (Table 4.2).
The correct muscular twitches after femoral and sciatic nerve electrolocation were easily found in all dogs in the PNB group. Cerebro-spinal fluid free flow in the hub of the needle was clearly seen in all dogs in the SPI group. No adverse reactions were observed after local anaesthetic injection in any case. None of the enrolled subjects in the PNB and SPI groups showed signs of nociception during the instrumentation and no complication associated to peripheral nerve blocks or spinal anaesthesia were observed. All dogs recovered their motor function after the off-set the local anaesthetic effect and no neurological deficit were observed during the 30 days follow-up period. During surgery, 13 out of 15 cases in the PNB group and 14 out of 15 cases in the SPI group, the HR and dMAP did not increase more than 20% compared to values registered at Tb, and did not require any intra-operative rescue analgesia. In two cases in the PNB group and in one case in the SPI group, one bolus of fentanyl (2 µg kg-1, IV) was
administered during the surgical procedure due to increases in HR or dMAP. In the FEN group, fentanyl administration was not associated with bradycardia (HR < than 40 beats/min), and in order to maintain
HR and dMAP within 20% of the values registered at Tb, the mean total
fentanyl rate infusion was 18.8 ± 6.3 µg kg h-1. No significant
points in any group. Differences regarding HR and dMAP among groups are represented in Fig. 4.8 and Fig. 4.9 respectively.
In the PNB and SPI groups, FE’ISO resulted significantly higher
(p<0.0001) at Tb, T0 and T5 than FE’ISO at T1/2 and at TEND (Fig. 4.10).
Moreover, FE’ISO was significantly higher (p<0.0001) at T1/2 and at TEND
in the FEN and CTR groups compared to PNB and SPI groups (Fig. 4.10).
Recovery median scores were significantly higher in the FEN group (p<0.0001) compared to all other groups, and they were 1 (1-3), 1 (1-4), 2 (2-4) and 1 (1-2) in PNB, SPI, FEN and CTR groups respectively. No differences were observed in the recovery scores among PNB, SPI and CTR groups (p=0.21).
The post-operative pain scores are represented in Fig. 4.11. At T0, the
pain score was significantly lower in the PNB group compared to SPI and FEN groups (p=0.006). Pain scores resulted significantly lower
(p=0.02) at T45 and T60 in the PNB group compared to SPI group (Fig.
4.11). No significant differences were present in the post-operative pain score between SPI and FEN groups (p=0.15).
None of the dogs in the PNB group required methadone administration during the one hour post-operative evaluation period. Postoperative methadone consumption was significantly higher in FEN group compared to SPI groups (p=0.0002). The median total
methadone dose required was 0.1 (0.1-0.35) mg kg-1 and 0.3 (0.2-0.4) mg
kg-1 in the SPI and FEN groups respectively.
The trends of the blood glucose level during the different time points and the comparison among groups are represented in the Fig. 4.12. Cortisol levels evaluated during the study are represented in the Fig. 4.13. Cortisol basal levels (TAr) were within the normal range reported
for dogs (Nelson 2006) in all groups, and did not increase significantly (p=0.054) after induction of anaesthesia in any group. In the FNT group, cortisol levels increased significantly from baseline at TEXT and
groups, it was revealed that in the FNT group, the serum cortisol level was significantly higher at TEXT and TPST compared to PNB, SPI and
CTR groups (p<0.0001). Cortisol levels did not increase significantly from the baseline (TAr) at TINT, TEXT and TPST in PNB, SPI and CTR
groups (Fig. 7).
In the two cases that required a single bolus of fentanyl as rescue analgesia, the cortisol level did not increase significantly (p=0.44) from the base line (TAr), and it was 3.1 ± 2.9, 2.7 ± 2.1, 2.0 ± 1.1, 1.4 ± 0.5 µg/
dL at TAr, TINT, TEXT and TPST respectively. The glucose level in these
two cases was 84.5 ± 0.7, 88.5 ± 3.5, 92.5 ± 2.1, 80.5 ± 3.5 mg/dL at TAr,
TINT, TEXT and TPST respectively without differences significative
* *
Table 4.2: Results of age, weight, gender, pre-anaesthetic behaviour score, type and duration of
procedure in the different groups.
* Dogs of the CTR group were significantly younger that dogs of the other groups (p<0.0001). Duration of the procedure was significantly shorter in the CTR group (p<0.0001) compared to the other groups.
Figure 4.8: HR at different time-points (Tb=baseline; T0=beginning of procedure; T5=5 minutes
after beginning of procedure; T1/2=half time of duration of procedure; TEND=end of procedure).
a: significant differences between FEN and SPI groups (p=0.02).
Figure 4.9: dMAP at different time-points (Tb=baseline; T0=beginning of procedure; T5=5
minutes after beginning of procedure; T1/2=half time of duration of procedure; TEND=end of
Figure 4.10: FE’ISO (vol%) at different time-points (Tb=baseline; T0=beginning of procedure;
T5=5 minutes after beginning of procedure; T1/2=half time of duration of procedure; TEND=end of
procedure).
a: significant differences between Tb, T0 and T5 compared to T1/2 and TEND in PNB and SPI
groups (p<0.0001).
b: significant differences between FEN and CTR groups compared to PNB and SPI groups
Figure 4.11: Post-operative pain scores at different time-points (T0=extubation; T15=15 minutes
after extubation; T30=30 minutes after extubation; T45=45 minutes after extubation; T60=60
minutes after extubation).
a: significant differences between PNB group compared to SPI and FEN groups (p=0.006). b: significant differences between PNB compared to SPI group (p=0.002)
Figure 4.12: blood glucose levels (mg/dl) at different time-points (TAr=arrival at the Hospital;
TINT=tracheal intubation; TEXT=tracheal extubation; TPST=one hour after extubation).
a: significant differences between FEN group compared to PNB, SPI and CTR groups (p=0.0002) b: significant differences between FEN group compared to PNB, SPI and CTR groups (p<0.0001)
Figure 4.13: Serum cortisol (µg/dl) at different time-points (TAr=arrival at the Hospital;
TINT=tracheal intubation; TEXT=tracheal extubation; TPST=one hour after extubation).
