C-reactive protein, glucose and iron concentrations are significantly altered in dogs undergoing open ovariohysterectomy or ovariectomy

C-reactive protein, glucose and iron concentrations are significantly altered in dogs undergoing... Background: There are relatively few studies about the canine surgical stress response, a sequence of events orches‑ trated by the body in response to a surgical trauma which is sometimes, as shown in human surgery, deleterious to the patient. There is a need to identify objective markers to quantify this response in order to estimate tissue trauma and use the markers as potential early indicators of surgical complications. The study objective was to investigate the surgical stress response, measured by C‑ reactive protein (CRP), glucose and iron serum concentrations, to gonadec‑ tomy in female dogs, and to compare the response to ovariohysterectomy (OHE) with the response to ovariectomy (OVE). A randomized clinical trial was performed on a sample of 42 female dogs, which were divided into two groups: one group underwent OHE, the other OVE. Results: Blood samples were collected immediately before surgery ( T0), and at 1 ( T1), 6 ( T6), and 24 ( T24) h after surgery, and serum frozen and stored at − 80 °C for later analysis. Upon thawing, the serum samples were subjected to measurement of CRP, glucose and iron concentration. Seventeen dogs in the OHE group and 19 dogs in the OVE group were included in the statistical analysis. There was a significant increase in glucose concentration at all time points compared with T0, and an increase of CRP at T6 and T24. Iron concentration was significantly decreased at T6 and T24. Differences between the two groups could not be detected for any of the three variables. Conclusions: The study showed that both OHE and OVE induce a moderate surgical stress response in female dogs, measured by CRP, glucose and iron. A difference between the surgical techniques could not be detected for any of the variables, and hence; with regards to the parameters studied recommendations of one procedure over the other cannot be made and preferred technique remains the surgeon’s choice. Keywords: C‑ reactive protein, Glucose, Iron, Ovariectomy, Ovariohysterectomy, Surgery, Surgical stress response *Correspondence: elena.moldal@nmbu.no Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Norway Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Moldal et al. Acta Vet Scand (2018) 60:32 Page 2 of 8 tested was: Surgery will cause significant increases of Background serum CRP and glucose and a decrease in serum iron- The stress response to surgery involves an array of physi - concentrations postoperatively, but to a lesser degree in ological events in the body, including endocrinological, the OVE compared to the OHE group. immunological, and hematological alterations leading to a catabolic state [1, 2]. Even though these functions are Methods beneficial in the acute survival situation, this response The study was approved by the Ethics and Research may in fact have negative effects on homeostasis and tis - Committee of the Department of Clinical Sciences of sue healing [3]. Companion Animals, Faculty of Veterinary Medicine, The surgical stress response is believed to be propor - University of Utrecht (DCSCA), the Netherlands. It was tional with the degree of tissue injury caused by the pro- performed as a prospective randomized clinical trial at cedure [4, 5]. It is therefore important to choose surgical the DCSCA between June 2006 and June 2007. Serum procedures that minimize the negative impact of surgery was stored at − 80 °C for a maximum of 4 years, and later on the body. Complications after elective surgery in dogs transported to the University of Copenhagen on dry ice and cats are not uncommon and have been reported to before analysis at the Central Laboratory, Department of include hemorrhage, surgical site inflammation or infec - Veterinary Clinical Sciences, University of Copenhagen, tion, and increased attention to the surgical site [6, 7]. Denmark, in November 2010. The laboratory analysis Female dogs are commonly neutered, most often by was performed double blind in one analytical run, in ran- open ovariohysterectomy (OHE) or ovariectomy (OVE). dom order, and unblinding did not take place until after Several authors argue that OVE should be the preferred statistical analysis of the data. Only the surgeon (MEP) method because of the belief that it is faster, safer, less knew what procedure was performed. invasive, and associated with fewer postoperative compli- cations [8–10]. Open OHE in dogs has previously been Study population shown to induce a significant, but short-lived neuroen - A total of 42 client-owned healthy intact bitches admit- docrine stress response [11]. Two previous studies by ted to the DCSCA for elective neutering were prospec- the authors comparing OVE and OHE failed to show dif- tively entered into the study. Of these, 12 bitches were ferences between the two methods with regards to pain mongrels and 30 were pure-bred. Oral consent was scores, time expenditure, and wound characteristics, as obtained from the owners before the dogs underwent a well as difference in the hemostatic stress response to thorough clinical examination to ensure that they were surgery [12, 13]. However, one recent study identified sig - healthy. Only dogs assigned to ASA category 1 (normal, nificant differences in postoperative C-reactive protein healthy animals) [21] were eligible for participation in (CRP) concentrations in three groups of dogs subjected the study, and all dogs went through their last estrus at to vasectomy, open OHE, or laparoscopic OHE [14]. least 6  weeks prior to presentation. Each dog was given CRP is an acute phase protein and a sensitive marker a body condition score (BCS) at admission, with a score of inflammation [15–17], and can be used to quantify of 1 being emaciated and 5 being obese. The dogs were the inflammatory response to different surgical proce - numbered consecutively at admission. Dogs were block dures in dogs [14]. Glucose is another biomarker com- randomized into one of two treatment groups, OVE or monly used to measure the stress response to surgery. OHE, after induction of anesthesia [12]. A study comparing dogs subjected to open OHE with dogs subjected to the laparoscopic counterpart identi- Anesthesia, surgery, and analgesia fied prolonged increases in glucose concentration in the An intravenous (IV) catheter was inserted in the cephalic open OHE group during the postoperative period [18]. vein. The dogs were given a premedication of 1  mg/ Glucose is also an independent risk factor for postopera- m medetomidine intravenously (Domitor, Pfizer Ani - tive wound infections in humans [19]. Hypoferremia is mal Health, USA, 1  mg/mL) and 4  mg/kg carprofen IV commonly seen after surgically induced inflammation in (Rimadyl, Pfizer Animal Health, USA, 50  mg/mL) and humans and is related to the extent of surgery [20]; how- anesthesia was induced with 1–2  mg/kg propofol IV ever, information about iron concentration after surgery (PropoVet, Abbott Laboratories, UK, 10  mg/mL) to in dogs is scarce. effect. The dogs were then intubated and anesthesia was The aims of this study were to measure CRP, iron and maintained with isoflurane (Isoflo, Abbott Laboratories, glucose serum concentration as markers of the surgical UK) in oxygen and air. Intermittent positive pressure stress response in dogs, and to test whether they differed ventilation (IPPV) was applied to ensure normocapnea between two commonly applied methods for surgical and the volume was regulated to keep end-tidal CO at neutering, of which one—OVE—has been claimed to be normal levels (4.5–5  kPa). All dogs were given 10  mL/ less traumatic by some authors [8–10]. The hypothesis Moldal et al. Acta Vet Scand (2018) 60:32 Page 3 of 8 kg/h Ringer’s lactate IV (Stereofundin, Iso; B, Germany) Other at maintenance rate throughout the course of anesthe- Hemostasis parameters and other variables including sia and surgery. Intraoperative monitoring consisted of blood loss, surgical time, surgical wound characteris- electrocardiogram (ECG), capnography, body tempera- tics, pain scores, and wound assessment scores were ture, and oxygen and vapor concentrations. In surgeries recorded and published in other studies [12, 13]. that lasted for more than one h, an additional dose cor- responding to half of the original administered dose of CRP medetomidine was administered IV. After surgery this CRP levels were analyzed using a turbidimetric immu- was antagonized with 2.5 µg/m atipamezole intramuscu- noassay (High Linearity CRP, Randox Laboratories larly (IM) (Antisedan, Pfizer Animal Health, USA, 5 mg/ Ltd., Crumlin, UK) performed on Advia 1800 Chemis- mL) [12]. try System (Siemens, Germany). Independently puri- All surgeries were performed by one experienced ECVS fied canine CRP was applied as calibrator (cat#8101, Diplomate (MEP) with the help of an assistant, using a Life Diagnostics, West Chester, PA, USA) and control standardized surgical protocol for both procedures. Both (TP-810CON, Tridelta, Kildare, Ireland). For complete OVE and OHE were carried out as open surgical proce- assay performance, please see validation conducted by dures. The OVE dogs had their ovaries removed through the laboratory performing the measurements [23, 24]. a smaller incision than the OHE dogs, which additionally Automated reflex dilution was applied when meas - had their uterus removed [12]. urement exceeded linear range, resulting in effective All dogs were hospitalized for 24–32  h postopera- working range up to 600 mg/L. No prozone effect were tively. 10  µg/kg buprenorphine (Buprecare, Animalcare observed up to 900 mg/L. Ltd, UK, 0.3 mg/mL) was administered IV approximately 40 m before injecting atipamezole and then given subcu- Glucose taneously (SC) every 6 h during the next 24 h. The rescue Glucose was measured with the reagent Glucose analgesia protocol consisted of administration of a higher Hexokinase/Glucose oxidase, including assay calibra- dose of buprenorphine 20  µg/kg SC to animals showing tor provided by manufacturer (Siemens, Germany) pain scores > 15 on a modified version of the Short Form performed on the Advia 1800 Chemistry System. (SF) of the Glasgow Composite Measure Pain Scale [22]. Imprecision was below 2%. Treatment at home consisted of 2 mg/kg carprofen orally every 12 h for an additional 2 days after discharge [12]. Iron Iron was measured by using the reagent Iron RGT KT Blood sampling D/S, including calibrator provided by manufacturer Immediately after anesthetic induction an IV jugular (Siemens, Germany) on the Advia 1800 Chemistry Sys- catheter was inserted and secured in place. Just before the tem. Imprecision was below 2%. skin incision (T0) and just before closure of the abdomi- nal incision (T1), and also at 6  h after T0 (T6), blood Statistical analysis samples were collected from this catheter after discard- Two dogs were excluded from the study, one because ing the first 5 mL of blood. The jugular catheter was then it was under treatment with phenobarbital for epilepsy, removed, and the 24-h blood sample (T24) was taken by the other because of unexpected complications dur- direct venipuncture of the contralateral jugular vein. For ing surgery which lengthened the procedure but were all samples, a total of 11  mL blood was collected in one not associated with the procedure per se. Also, because serum tube and two 3.2% citrate tubes, in that order. For four serum samples were stored in a different freezer T0, 10 additional mL blood was collected in heparin and for a period of time, one sample from the OVE group EDTA for biochemistry and hematology, to confirm the and three samples from the OHE group were discarded. animal’s health before enrolment in the project. The fol - Thus, results from 36 dogs, 17 in the OHE group and lowing variables were analyzed: BUN (blood urea nitro- 19 in the OVE group, were included in the statistical gen), serum creatinine, alkaline phosphatase, bile acids, analysis. All statistical analyses were performed using total plasma calcium, phosphorus, sodium, potassium, the statistical software package Stata version 11 (Stata- hematocrit, total leucocytes, and platelets. corp, College Station, USA). Three separate regression All serum tubes were left in room temperature and analyses were performed; one for each of the outcome centrifuged after 1  h at 4  °C at 1006g for 10  min before variables CRP, glucose and serum iron concentra- the serum was separated and placed directly in a − 80  °C tions. The explanatory variables were treatment group freezer for later analysis at the Department of Veterinary (OVE or OHE) and time [0 (= baseline), 1, 6, 24  h] in Clinical Sciences. Moldal et al. Acta Vet Scand (2018) 60:32 Page 4 of 8 all models. Variables were initially evaluated for corre- lations between time points. Observations within each dog through time were not independent of each other. Therefore linear mixed regression models, including random effects for dog, were applied to detect differ - ences between the treatment groups and between time points for each of the outcome variables. The overall effect of the categorical variable time was tested using likelihood ratio (LR) tests. The level of statistical signifi - cance was set to P < 0.05. The assumption of normally distributed residuals was assessed using normal quan- tile plots at the dog level. Results The mean age of participating dogs in the sample was 3.4  years, range 6  months to 10  years, and the mean weight 25  kg, range 12–36  kg. The groups did not differ with regards to age, body weight, body condition score, and surgical time [12]. Preoperative biochemical and hematological profiles in the dogs were within the refer - ence intervals of the DCSCA. None of the dogs had pain scores > 15 and thus, rescue analgesia was not indicated in any of the animals. CRP, glucose and iron Mean and standard deviation for CRP, glucose and iron serum concentrations by time and group are presented in Table 1. The baseline (T0) values did not differ signifi - cantly between the groups for any of the three variables. Based on the observed correlations, an exchangeable cor- Fig. 1 Mean CRP (a), glucose (b), and iron (c) concentrations for the relation structure between time points was assumed for OHE and OVE group at each time point T0, T1, T6 and T24. There was glucose and CRP, and a first-order autoregressive for iron no difference between groups for any of the parameters but the concentration. The reported effects of treatment group statistically significant changes from T0 are marked with asterisks and time are based on output from the three regres- sion models for CRP, glucose and iron (Model output Table 1 Mean and standard deviation (SD) of CRP, glucose, and iron serum concentrations for dogs in the OHE and OVE group Variable Time OHE mean SD OVE mean SD Reference interval CRP (mg/L) 0 2.7 5.7 2.5 5.3 0.4–15.9 1 2.9 5.7 2.3 4.8 6 11.4 10.7 13.2 9.8 24 57.6 38.4 58.3 25.0 Glucose (mmol/L) 0 5.9 0.7 6.1 1.0 3.9–6.6 1 6.4 0.9 6.4 0.9 6 6.5 0.5 6.8 0.7 24 6.3 0.6 6.5 0.5 Iron (µmol/L) 0 22.4 6.5 22.4 7.5 5.4–32.2 1 22.7 6.3 21.6 6.4 6 16.1 6.2 13.8 6.0 24 14.5 7.3 14.6 5.9 Moldal et al. Acta Vet Scand (2018) 60:32 Page 5 of 8 available from the first author by request). CRP (Fig.  1a) same phenomenon has been identified in dogs [14]. The was increased at T6 and T24 (P < 0.001) for both groups. results from the current study serve to indicate that tis- There was no significant difference in CRP between sue trauma, as measured by CRP, is comparable for open groups (P = 0.92). The glucose concentration (Fig.  1b) OVE and OHE. was higher than baseline (T0) at all time points (LR test The glucose concentration significantly increased at of group; P = 0.004), but no difference between groups T1 and T6, but slightly decreased again at T24; how- was detected (P = 0.27). Iron concentration (Fig.  1c) was ever, the difference from T0 to T24 was still statistically decreased at T6 and T24 compared to baseline (P < 0.001 significant. There was no difference between the two for both), with no difference between groups (P = 0.68). groups. Blood glucose concentration is a useful measure Residuals were approximately normally distributed for all of surgical stress in dogs [18], and has been identified as three models when assessed at the dog-level using nor- an independent risk factor for infection after surgery in mal quantile plots. The random dog-effect was highly sig - humans [19, 29]. Hyperglycemia has deleterious effects nificant for all three variables. on macrophage and neutrophil function [39], and this may explain why human patients suffering from diabe - Discussion tes mellitus are twice as likely to develop a post-operative Both OHE and OVE induced significant postoperative infection compared to normoglycemic individuals [40, changes in CRP, glucose and iron concentrations. The 41]. The pathophysiology behind postoperative hypergly - hypothesis that OVE would cause a less marked stress cemia is partly induction of a hyperglycemic response by response could however not be supported, which corrob- cortisol and growth hormone and partly insulin resist- orates the authors’ two previous studies comparing OVE ance and inhibition of insulin secretion, all induced by and OHE [12, 13]. The detection of increased CRP after the neuroendocrine and metabolic stress response to surgery is in accordance with previous human and canine surgery [5]. Glucose concentration has also been shown studies [14–17, 25–28]. Increased glucose concentration to have predictive value on the outcome in critically ill perioperatively has also been registered in both species human patients [42]. In a study by Benson et  al. [11], [11, 18, 19, 29]. glucose was found to be elevated after anesthesia and Decreased iron concentration has been reported both surgery (OHE) in dogs. The increasing glucose concen - after soft tissue and orthopedic surgery in humans [20, tration up to T6 corroborates a previous study on OHE 30], and the magnitude of this decrease differs with sur - in dogs [18]. In a study by Hardie et al. [43], 50% of dogs gical invasiveness [20]. Information about iron concen- with sepsis that developed high glucose concentrations tration in dogs after surgery is scarce, but unpublished postoperatively died, whereas mortality in the group with observations by the authors indicate decreased concen- normal glucose concentration was 14%. The difference trations after both skin-, abdominal- and orthopedic sur- was, however, not statistically significant (P = 0.08) [43]. gery compared with pre-operative values in dogs. The link between high glucose concentration and mor - CRP is a major acute phase protein in dogs and the bidity is not completely understood, but it has been sug- results expectedly indicate that a moderate inflammatory gested that the responsiveness of leukocytes stimulated response occurs after both OVE and OHE. CRP has been with inflammatory mediators is inversely correlated with shown to be a sensitive marker of inflammation and fur - indices of in  vivo glycemic control in humans [39]. As a ther has the ability to distinguish inflammatory states as minor study limitation it should be noted that time of a result of neoplasia, immune-mediated disease, surgery, postoperative feeding is not available for the dogs in the and infections [14, 31–35]. It has been argued that CRP study. Also, because the postoperative glucose concentra- should be part of routine diagnostic testing because of its tion was in the upper end of, and not outside, the refer- higher sensitivity than WBC [36, 37]. CRP can increase ence interval for dogs in our study, a clinical relevance is up to 95 times as a result of surgery [26], and this increase considered unlikely. Nevertheless, it seems that OHE and is related to the degree of tissue injury in dogs [14, 26]. OVE induce increased glucose concentration to a compa- u Th s, CRP can be used to reflect the degree of surgical rable extent. trauma [14]. In our study CRP increased approximately The iron concentration decreased to a similar degree 20-fold from T0 to T24. There was no difference between in both groups after surgery, at T6 and T24. An anemic groups. OHE has previously been shown to cause mod- state that resembles anemia of chronic disease com- erately elevated CRP in dogs [16, 26]; however, to a lesser monly occurs in humans after surgery [20, 30], and can degree than more invasive surgery like orthopedic sur- take up to 6  weeks to normalize [30]. This was previ - gery [26]. In a study of humans, a smaller elevation of ously believed to be purely due to blood loss; however, CRP was detected after laparoscopic hysterectomy com- iron supplementation after orthopedic surgery has no pared to the open abdominal procedure [38], and the major effect on erythropoiesis [44, 45]. Research in mice Moldal et al. Acta Vet Scand (2018) 60:32 Page 6 of 8 indicates that hypoferremia is mediated by interleukin 6 The results from the current study show that open (IL-6) because it induces synthesis of the iron regulatory OVE and OHE provoke a moderate surgical stress hormone hepcidin, an acute phase protein in humans response, as measured by CRP, glucose and iron con- [46, 47]. Transferrin, an iron binding transporter protein, centration, of similar magnitude, likely because the is also a negative acute phase protein in dogs [31]. There two methods are too similar in surgical invasiveness are great similarities between dogs and humans in iron to detect subtle differences. Laparascopic techniques metabolism [48], and the mechanisms triggered postop- may confer advantages over OHE and OVE in limiting eratively are likely to be similar as well. The iron concen - inflammation and pain in the postoperative period [14, tration decreased to a similar extent in both groups. 18, 53–55]. It should be noted that several factors may influence the surgical stress response. Stress caused by hospitaliza- tion is commonly seen in dogs and may exacerbate the Conclusions endocrine responses to surgery [18]. Care must be taken The study showed that OHE and OVE induce a surgical to avoid stress in surgical patients in order to minimize stress response with postoperative increases in glucose the catabolic events mediated by the stress response. This concentration and CRP, and a decrease in iron concen- can in part be done with sedative and anesthetic drugs. tration. No significant difference between the OHE and In this study, medetomidine was used for premedica- OVE group could be detected with regards to the param- tion. Medetomidine has been shown to obtund the sur- eters measured, and a recommendation of one procedure gical stress response by preventing the catecholamine over the other can therefore not be made based on the response induced by OHE [11], and could therefore findings of this study. have affected the glucose concentration to some degree. There is no evidence in the literature to say that medeto - Abbreviations midine has an anti-inflammatory effect, and hence, an CRP: C‑reactive protein; DCSCA: The Department of Clinical Sciences of influence on CRP and iron concentration is considered Companion Animals, Faculty of Veterinary Medicine, University of Utrecht; IL‑6: interleukin 6; NSAIDs: non‑steroidal anti‑inflammatory drugs; OHE: ovariohys‑ unlikely. One could argue that the use of non-steroidal terectomy; OVE: ovariectomy. anti-inflammatory drugs (NSAIDs) such as carprofen would limit the inflammatory response to surgery; how - Authors’ contributions ERM collected data, stored samples, and participated in the laboratory ever, it is believed that NSAIDs do not directly block analyses. She was involved in statistical analysis of the data and responsible the production of IL-6 [49], which is proposed to be the for manuscript preparation. MKH was responsible for the choice of laboratory main inducer of CRP [26, 50]. Also, it has previously been variables. He performed the laboratory analyses and interpreted them, and was a major contributor in writing the manuscript. MEP was responsible for shown that CRP and iron as inflammatory markers are the design of the study and the acquisition of patients. She performed all sur‑ not affected by NSAID administration in humans [51], geries and was a major contributor in writing the manuscript. AN performed and neither meloxicam nor carprofen administration the statistical analyses and was responsible for the interpretation of these. She wrote the statistics section of the manuscript and contributed to other caused lower postoperative concentrations of CRP in a aspects of manuscript preparation. JK was responsible for the study design study of OHE in dogs [28]. Also, since carprofen admin- and the acquisition of patients, as well as sample collection, handling, and istration would impact the two groups to a similar extent, interpretation of results. He was a major contributor in writing the manuscript. All authors read and approved the final manuscript. we consider it a minor limitation to the study. The effects of stress and administration of anesthetic and analgesic Author details drugs are also assumed to be similar for both groups, but Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Nor‑ it cannot be excluded that the drugs have masked the sur- way. Department of Veterinary Clinical Sciences, Faculty of Health and Medi‑ gical stress response and hence masked a potential small 3 cal Sciences, University of Copenhagen, Copenhagen, Denmark. Department difference between groups. A previous study has shown of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands. Department of Production higher CRP concentrations after canine OHE performed Animal Clinical Sciences, Faculty of Veterinary Medicine and Biosciences, by inexperienced surgeons [27]; however, since we used 5 Norwegian University of Life Sciences, Oslo, Norway. Hill’s Pet Nutrition Inc, the same, experienced surgeon for all procedures, this Topeka, KS, USA. is not relevant for the current study. The dogs were only Acknowledgements followed for 24 h, and a follow-up to assess wound heal- The authors wish to thank Professor Thomas Eriksen for valuable input during ing or inflammatory complications was not carried out. data collection, analysis, and manuscript preparation. In humans, increased perioperative concentrations of Competing interests glucose and CRP have been described as risk factors for The authors declare that they have no competing interests. postoperative infections [19, 52]. A study with longer Availability of data and materials follow-up of the animals with regards to complications The datasets used and analyzed during the current study are available from resulting from surgery would have been of value. the corresponding author on reasonable request. Moldal et al. 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Hepcidin, scalpel‑assisted laparoscopy compared with median celiotomy and liga‑ a putative mediator of anemia of inflammation, is a type II acute ‑phase tion in dogs. Vet Surg. 2005;34:273–82. protein. Blood. 2003;101:2461–3. 54. Davidson EB, Moll HD, Payton ME. Comparison of laparoscopic ovariohys‑ 47. Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, et al. IL‑6 terectomy and ovariohysterectomy in dogs. Vet Surg. 2004;33:62–9. mediates hypoferremia of inflammation by inducing the synthesis of the 55. Culp WTN, Mayhew PD, Brown DC. The effect of laparoscopic versus iron regulatory hormone hepcidin. J Clin Invest. 2004;113:1271–6. open ovariectomy on postsurgical activity in small dogs. Vet Surg. 48. Finch CA, Hegsted M, Kinney TD, Thomas ED, Rath CE, Haskins D, 2009;38:811–7. et al. Iron metabolism: the pathophysiology of iron storage. Blood. 1950;5:983–1008. Ready to submit your research ? 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C-reactive protein, glucose and iron concentrations are significantly altered in dogs undergoing open ovariohysterectomy or ovariectomy

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Medicine & Public Health; Veterinary Medicine/Veterinary Science; Animal Anatomy / Morphology / Histology
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Abstract

Background: There are relatively few studies about the canine surgical stress response, a sequence of events orches‑ trated by the body in response to a surgical trauma which is sometimes, as shown in human surgery, deleterious to the patient. There is a need to identify objective markers to quantify this response in order to estimate tissue trauma and use the markers as potential early indicators of surgical complications. The study objective was to investigate the surgical stress response, measured by C‑ reactive protein (CRP), glucose and iron serum concentrations, to gonadec‑ tomy in female dogs, and to compare the response to ovariohysterectomy (OHE) with the response to ovariectomy (OVE). A randomized clinical trial was performed on a sample of 42 female dogs, which were divided into two groups: one group underwent OHE, the other OVE. Results: Blood samples were collected immediately before surgery ( T0), and at 1 ( T1), 6 ( T6), and 24 ( T24) h after surgery, and serum frozen and stored at − 80 °C for later analysis. Upon thawing, the serum samples were subjected to measurement of CRP, glucose and iron concentration. Seventeen dogs in the OHE group and 19 dogs in the OVE group were included in the statistical analysis. There was a significant increase in glucose concentration at all time points compared with T0, and an increase of CRP at T6 and T24. Iron concentration was significantly decreased at T6 and T24. Differences between the two groups could not be detected for any of the three variables. Conclusions: The study showed that both OHE and OVE induce a moderate surgical stress response in female dogs, measured by CRP, glucose and iron. A difference between the surgical techniques could not be detected for any of the variables, and hence; with regards to the parameters studied recommendations of one procedure over the other cannot be made and preferred technique remains the surgeon’s choice. Keywords: C‑ reactive protein, Glucose, Iron, Ovariectomy, Ovariohysterectomy, Surgery, Surgical stress response *Correspondence: elena.moldal@nmbu.no Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Norway Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Moldal et al. Acta Vet Scand (2018) 60:32 Page 2 of 8 tested was: Surgery will cause significant increases of Background serum CRP and glucose and a decrease in serum iron- The stress response to surgery involves an array of physi - concentrations postoperatively, but to a lesser degree in ological events in the body, including endocrinological, the OVE compared to the OHE group. immunological, and hematological alterations leading to a catabolic state [1, 2]. Even though these functions are Methods beneficial in the acute survival situation, this response The study was approved by the Ethics and Research may in fact have negative effects on homeostasis and tis - Committee of the Department of Clinical Sciences of sue healing [3]. Companion Animals, Faculty of Veterinary Medicine, The surgical stress response is believed to be propor - University of Utrecht (DCSCA), the Netherlands. It was tional with the degree of tissue injury caused by the pro- performed as a prospective randomized clinical trial at cedure [4, 5]. It is therefore important to choose surgical the DCSCA between June 2006 and June 2007. Serum procedures that minimize the negative impact of surgery was stored at − 80 °C for a maximum of 4 years, and later on the body. Complications after elective surgery in dogs transported to the University of Copenhagen on dry ice and cats are not uncommon and have been reported to before analysis at the Central Laboratory, Department of include hemorrhage, surgical site inflammation or infec - Veterinary Clinical Sciences, University of Copenhagen, tion, and increased attention to the surgical site [6, 7]. Denmark, in November 2010. The laboratory analysis Female dogs are commonly neutered, most often by was performed double blind in one analytical run, in ran- open ovariohysterectomy (OHE) or ovariectomy (OVE). dom order, and unblinding did not take place until after Several authors argue that OVE should be the preferred statistical analysis of the data. Only the surgeon (MEP) method because of the belief that it is faster, safer, less knew what procedure was performed. invasive, and associated with fewer postoperative compli- cations [8–10]. Open OHE in dogs has previously been Study population shown to induce a significant, but short-lived neuroen - A total of 42 client-owned healthy intact bitches admit- docrine stress response [11]. Two previous studies by ted to the DCSCA for elective neutering were prospec- the authors comparing OVE and OHE failed to show dif- tively entered into the study. Of these, 12 bitches were ferences between the two methods with regards to pain mongrels and 30 were pure-bred. Oral consent was scores, time expenditure, and wound characteristics, as obtained from the owners before the dogs underwent a well as difference in the hemostatic stress response to thorough clinical examination to ensure that they were surgery [12, 13]. However, one recent study identified sig - healthy. Only dogs assigned to ASA category 1 (normal, nificant differences in postoperative C-reactive protein healthy animals) [21] were eligible for participation in (CRP) concentrations in three groups of dogs subjected the study, and all dogs went through their last estrus at to vasectomy, open OHE, or laparoscopic OHE [14]. least 6  weeks prior to presentation. Each dog was given CRP is an acute phase protein and a sensitive marker a body condition score (BCS) at admission, with a score of inflammation [15–17], and can be used to quantify of 1 being emaciated and 5 being obese. The dogs were the inflammatory response to different surgical proce - numbered consecutively at admission. Dogs were block dures in dogs [14]. Glucose is another biomarker com- randomized into one of two treatment groups, OVE or monly used to measure the stress response to surgery. OHE, after induction of anesthesia [12]. A study comparing dogs subjected to open OHE with dogs subjected to the laparoscopic counterpart identi- Anesthesia, surgery, and analgesia fied prolonged increases in glucose concentration in the An intravenous (IV) catheter was inserted in the cephalic open OHE group during the postoperative period [18]. vein. The dogs were given a premedication of 1  mg/ Glucose is also an independent risk factor for postopera- m medetomidine intravenously (Domitor, Pfizer Ani - tive wound infections in humans [19]. Hypoferremia is mal Health, USA, 1  mg/mL) and 4  mg/kg carprofen IV commonly seen after surgically induced inflammation in (Rimadyl, Pfizer Animal Health, USA, 50  mg/mL) and humans and is related to the extent of surgery [20]; how- anesthesia was induced with 1–2  mg/kg propofol IV ever, information about iron concentration after surgery (PropoVet, Abbott Laboratories, UK, 10  mg/mL) to in dogs is scarce. effect. The dogs were then intubated and anesthesia was The aims of this study were to measure CRP, iron and maintained with isoflurane (Isoflo, Abbott Laboratories, glucose serum concentration as markers of the surgical UK) in oxygen and air. Intermittent positive pressure stress response in dogs, and to test whether they differed ventilation (IPPV) was applied to ensure normocapnea between two commonly applied methods for surgical and the volume was regulated to keep end-tidal CO at neutering, of which one—OVE—has been claimed to be normal levels (4.5–5  kPa). All dogs were given 10  mL/ less traumatic by some authors [8–10]. The hypothesis Moldal et al. Acta Vet Scand (2018) 60:32 Page 3 of 8 kg/h Ringer’s lactate IV (Stereofundin, Iso; B, Germany) Other at maintenance rate throughout the course of anesthe- Hemostasis parameters and other variables including sia and surgery. Intraoperative monitoring consisted of blood loss, surgical time, surgical wound characteris- electrocardiogram (ECG), capnography, body tempera- tics, pain scores, and wound assessment scores were ture, and oxygen and vapor concentrations. In surgeries recorded and published in other studies [12, 13]. that lasted for more than one h, an additional dose cor- responding to half of the original administered dose of CRP medetomidine was administered IV. After surgery this CRP levels were analyzed using a turbidimetric immu- was antagonized with 2.5 µg/m atipamezole intramuscu- noassay (High Linearity CRP, Randox Laboratories larly (IM) (Antisedan, Pfizer Animal Health, USA, 5 mg/ Ltd., Crumlin, UK) performed on Advia 1800 Chemis- mL) [12]. try System (Siemens, Germany). Independently puri- All surgeries were performed by one experienced ECVS fied canine CRP was applied as calibrator (cat#8101, Diplomate (MEP) with the help of an assistant, using a Life Diagnostics, West Chester, PA, USA) and control standardized surgical protocol for both procedures. Both (TP-810CON, Tridelta, Kildare, Ireland). For complete OVE and OHE were carried out as open surgical proce- assay performance, please see validation conducted by dures. The OVE dogs had their ovaries removed through the laboratory performing the measurements [23, 24]. a smaller incision than the OHE dogs, which additionally Automated reflex dilution was applied when meas - had their uterus removed [12]. urement exceeded linear range, resulting in effective All dogs were hospitalized for 24–32  h postopera- working range up to 600 mg/L. No prozone effect were tively. 10  µg/kg buprenorphine (Buprecare, Animalcare observed up to 900 mg/L. Ltd, UK, 0.3 mg/mL) was administered IV approximately 40 m before injecting atipamezole and then given subcu- Glucose taneously (SC) every 6 h during the next 24 h. The rescue Glucose was measured with the reagent Glucose analgesia protocol consisted of administration of a higher Hexokinase/Glucose oxidase, including assay calibra- dose of buprenorphine 20  µg/kg SC to animals showing tor provided by manufacturer (Siemens, Germany) pain scores > 15 on a modified version of the Short Form performed on the Advia 1800 Chemistry System. (SF) of the Glasgow Composite Measure Pain Scale [22]. Imprecision was below 2%. Treatment at home consisted of 2 mg/kg carprofen orally every 12 h for an additional 2 days after discharge [12]. Iron Iron was measured by using the reagent Iron RGT KT Blood sampling D/S, including calibrator provided by manufacturer Immediately after anesthetic induction an IV jugular (Siemens, Germany) on the Advia 1800 Chemistry Sys- catheter was inserted and secured in place. Just before the tem. Imprecision was below 2%. skin incision (T0) and just before closure of the abdomi- nal incision (T1), and also at 6  h after T0 (T6), blood Statistical analysis samples were collected from this catheter after discard- Two dogs were excluded from the study, one because ing the first 5 mL of blood. The jugular catheter was then it was under treatment with phenobarbital for epilepsy, removed, and the 24-h blood sample (T24) was taken by the other because of unexpected complications dur- direct venipuncture of the contralateral jugular vein. For ing surgery which lengthened the procedure but were all samples, a total of 11  mL blood was collected in one not associated with the procedure per se. Also, because serum tube and two 3.2% citrate tubes, in that order. For four serum samples were stored in a different freezer T0, 10 additional mL blood was collected in heparin and for a period of time, one sample from the OVE group EDTA for biochemistry and hematology, to confirm the and three samples from the OHE group were discarded. animal’s health before enrolment in the project. The fol - Thus, results from 36 dogs, 17 in the OHE group and lowing variables were analyzed: BUN (blood urea nitro- 19 in the OVE group, were included in the statistical gen), serum creatinine, alkaline phosphatase, bile acids, analysis. All statistical analyses were performed using total plasma calcium, phosphorus, sodium, potassium, the statistical software package Stata version 11 (Stata- hematocrit, total leucocytes, and platelets. corp, College Station, USA). Three separate regression All serum tubes were left in room temperature and analyses were performed; one for each of the outcome centrifuged after 1  h at 4  °C at 1006g for 10  min before variables CRP, glucose and serum iron concentra- the serum was separated and placed directly in a − 80  °C tions. The explanatory variables were treatment group freezer for later analysis at the Department of Veterinary (OVE or OHE) and time [0 (= baseline), 1, 6, 24  h] in Clinical Sciences. Moldal et al. Acta Vet Scand (2018) 60:32 Page 4 of 8 all models. Variables were initially evaluated for corre- lations between time points. Observations within each dog through time were not independent of each other. Therefore linear mixed regression models, including random effects for dog, were applied to detect differ - ences between the treatment groups and between time points for each of the outcome variables. The overall effect of the categorical variable time was tested using likelihood ratio (LR) tests. The level of statistical signifi - cance was set to P < 0.05. The assumption of normally distributed residuals was assessed using normal quan- tile plots at the dog level. Results The mean age of participating dogs in the sample was 3.4  years, range 6  months to 10  years, and the mean weight 25  kg, range 12–36  kg. The groups did not differ with regards to age, body weight, body condition score, and surgical time [12]. Preoperative biochemical and hematological profiles in the dogs were within the refer - ence intervals of the DCSCA. None of the dogs had pain scores > 15 and thus, rescue analgesia was not indicated in any of the animals. CRP, glucose and iron Mean and standard deviation for CRP, glucose and iron serum concentrations by time and group are presented in Table 1. The baseline (T0) values did not differ signifi - cantly between the groups for any of the three variables. Based on the observed correlations, an exchangeable cor- Fig. 1 Mean CRP (a), glucose (b), and iron (c) concentrations for the relation structure between time points was assumed for OHE and OVE group at each time point T0, T1, T6 and T24. There was glucose and CRP, and a first-order autoregressive for iron no difference between groups for any of the parameters but the concentration. The reported effects of treatment group statistically significant changes from T0 are marked with asterisks and time are based on output from the three regres- sion models for CRP, glucose and iron (Model output Table 1 Mean and standard deviation (SD) of CRP, glucose, and iron serum concentrations for dogs in the OHE and OVE group Variable Time OHE mean SD OVE mean SD Reference interval CRP (mg/L) 0 2.7 5.7 2.5 5.3 0.4–15.9 1 2.9 5.7 2.3 4.8 6 11.4 10.7 13.2 9.8 24 57.6 38.4 58.3 25.0 Glucose (mmol/L) 0 5.9 0.7 6.1 1.0 3.9–6.6 1 6.4 0.9 6.4 0.9 6 6.5 0.5 6.8 0.7 24 6.3 0.6 6.5 0.5 Iron (µmol/L) 0 22.4 6.5 22.4 7.5 5.4–32.2 1 22.7 6.3 21.6 6.4 6 16.1 6.2 13.8 6.0 24 14.5 7.3 14.6 5.9 Moldal et al. Acta Vet Scand (2018) 60:32 Page 5 of 8 available from the first author by request). CRP (Fig.  1a) same phenomenon has been identified in dogs [14]. The was increased at T6 and T24 (P < 0.001) for both groups. results from the current study serve to indicate that tis- There was no significant difference in CRP between sue trauma, as measured by CRP, is comparable for open groups (P = 0.92). The glucose concentration (Fig.  1b) OVE and OHE. was higher than baseline (T0) at all time points (LR test The glucose concentration significantly increased at of group; P = 0.004), but no difference between groups T1 and T6, but slightly decreased again at T24; how- was detected (P = 0.27). Iron concentration (Fig.  1c) was ever, the difference from T0 to T24 was still statistically decreased at T6 and T24 compared to baseline (P < 0.001 significant. There was no difference between the two for both), with no difference between groups (P = 0.68). groups. Blood glucose concentration is a useful measure Residuals were approximately normally distributed for all of surgical stress in dogs [18], and has been identified as three models when assessed at the dog-level using nor- an independent risk factor for infection after surgery in mal quantile plots. The random dog-effect was highly sig - humans [19, 29]. Hyperglycemia has deleterious effects nificant for all three variables. on macrophage and neutrophil function [39], and this may explain why human patients suffering from diabe - Discussion tes mellitus are twice as likely to develop a post-operative Both OHE and OVE induced significant postoperative infection compared to normoglycemic individuals [40, changes in CRP, glucose and iron concentrations. The 41]. The pathophysiology behind postoperative hypergly - hypothesis that OVE would cause a less marked stress cemia is partly induction of a hyperglycemic response by response could however not be supported, which corrob- cortisol and growth hormone and partly insulin resist- orates the authors’ two previous studies comparing OVE ance and inhibition of insulin secretion, all induced by and OHE [12, 13]. The detection of increased CRP after the neuroendocrine and metabolic stress response to surgery is in accordance with previous human and canine surgery [5]. Glucose concentration has also been shown studies [14–17, 25–28]. Increased glucose concentration to have predictive value on the outcome in critically ill perioperatively has also been registered in both species human patients [42]. In a study by Benson et  al. [11], [11, 18, 19, 29]. glucose was found to be elevated after anesthesia and Decreased iron concentration has been reported both surgery (OHE) in dogs. The increasing glucose concen - after soft tissue and orthopedic surgery in humans [20, tration up to T6 corroborates a previous study on OHE 30], and the magnitude of this decrease differs with sur - in dogs [18]. In a study by Hardie et al. [43], 50% of dogs gical invasiveness [20]. Information about iron concen- with sepsis that developed high glucose concentrations tration in dogs after surgery is scarce, but unpublished postoperatively died, whereas mortality in the group with observations by the authors indicate decreased concen- normal glucose concentration was 14%. The difference trations after both skin-, abdominal- and orthopedic sur- was, however, not statistically significant (P = 0.08) [43]. gery compared with pre-operative values in dogs. The link between high glucose concentration and mor - CRP is a major acute phase protein in dogs and the bidity is not completely understood, but it has been sug- results expectedly indicate that a moderate inflammatory gested that the responsiveness of leukocytes stimulated response occurs after both OVE and OHE. CRP has been with inflammatory mediators is inversely correlated with shown to be a sensitive marker of inflammation and fur - indices of in  vivo glycemic control in humans [39]. As a ther has the ability to distinguish inflammatory states as minor study limitation it should be noted that time of a result of neoplasia, immune-mediated disease, surgery, postoperative feeding is not available for the dogs in the and infections [14, 31–35]. It has been argued that CRP study. Also, because the postoperative glucose concentra- should be part of routine diagnostic testing because of its tion was in the upper end of, and not outside, the refer- higher sensitivity than WBC [36, 37]. CRP can increase ence interval for dogs in our study, a clinical relevance is up to 95 times as a result of surgery [26], and this increase considered unlikely. Nevertheless, it seems that OHE and is related to the degree of tissue injury in dogs [14, 26]. OVE induce increased glucose concentration to a compa- u Th s, CRP can be used to reflect the degree of surgical rable extent. trauma [14]. In our study CRP increased approximately The iron concentration decreased to a similar degree 20-fold from T0 to T24. There was no difference between in both groups after surgery, at T6 and T24. An anemic groups. OHE has previously been shown to cause mod- state that resembles anemia of chronic disease com- erately elevated CRP in dogs [16, 26]; however, to a lesser monly occurs in humans after surgery [20, 30], and can degree than more invasive surgery like orthopedic sur- take up to 6  weeks to normalize [30]. This was previ - gery [26]. In a study of humans, a smaller elevation of ously believed to be purely due to blood loss; however, CRP was detected after laparoscopic hysterectomy com- iron supplementation after orthopedic surgery has no pared to the open abdominal procedure [38], and the major effect on erythropoiesis [44, 45]. Research in mice Moldal et al. Acta Vet Scand (2018) 60:32 Page 6 of 8 indicates that hypoferremia is mediated by interleukin 6 The results from the current study show that open (IL-6) because it induces synthesis of the iron regulatory OVE and OHE provoke a moderate surgical stress hormone hepcidin, an acute phase protein in humans response, as measured by CRP, glucose and iron con- [46, 47]. Transferrin, an iron binding transporter protein, centration, of similar magnitude, likely because the is also a negative acute phase protein in dogs [31]. There two methods are too similar in surgical invasiveness are great similarities between dogs and humans in iron to detect subtle differences. Laparascopic techniques metabolism [48], and the mechanisms triggered postop- may confer advantages over OHE and OVE in limiting eratively are likely to be similar as well. The iron concen - inflammation and pain in the postoperative period [14, tration decreased to a similar extent in both groups. 18, 53–55]. It should be noted that several factors may influence the surgical stress response. Stress caused by hospitaliza- tion is commonly seen in dogs and may exacerbate the Conclusions endocrine responses to surgery [18]. Care must be taken The study showed that OHE and OVE induce a surgical to avoid stress in surgical patients in order to minimize stress response with postoperative increases in glucose the catabolic events mediated by the stress response. This concentration and CRP, and a decrease in iron concen- can in part be done with sedative and anesthetic drugs. tration. No significant difference between the OHE and In this study, medetomidine was used for premedica- OVE group could be detected with regards to the param- tion. Medetomidine has been shown to obtund the sur- eters measured, and a recommendation of one procedure gical stress response by preventing the catecholamine over the other can therefore not be made based on the response induced by OHE [11], and could therefore findings of this study. have affected the glucose concentration to some degree. There is no evidence in the literature to say that medeto - Abbreviations midine has an anti-inflammatory effect, and hence, an CRP: C‑reactive protein; DCSCA: The Department of Clinical Sciences of influence on CRP and iron concentration is considered Companion Animals, Faculty of Veterinary Medicine, University of Utrecht; IL‑6: interleukin 6; NSAIDs: non‑steroidal anti‑inflammatory drugs; OHE: ovariohys‑ unlikely. One could argue that the use of non-steroidal terectomy; OVE: ovariectomy. anti-inflammatory drugs (NSAIDs) such as carprofen would limit the inflammatory response to surgery; how - Authors’ contributions ERM collected data, stored samples, and participated in the laboratory ever, it is believed that NSAIDs do not directly block analyses. She was involved in statistical analysis of the data and responsible the production of IL-6 [49], which is proposed to be the for manuscript preparation. MKH was responsible for the choice of laboratory main inducer of CRP [26, 50]. Also, it has previously been variables. He performed the laboratory analyses and interpreted them, and was a major contributor in writing the manuscript. MEP was responsible for shown that CRP and iron as inflammatory markers are the design of the study and the acquisition of patients. She performed all sur‑ not affected by NSAID administration in humans [51], geries and was a major contributor in writing the manuscript. AN performed and neither meloxicam nor carprofen administration the statistical analyses and was responsible for the interpretation of these. She wrote the statistics section of the manuscript and contributed to other caused lower postoperative concentrations of CRP in a aspects of manuscript preparation. JK was responsible for the study design study of OHE in dogs [28]. Also, since carprofen admin- and the acquisition of patients, as well as sample collection, handling, and istration would impact the two groups to a similar extent, interpretation of results. He was a major contributor in writing the manuscript. All authors read and approved the final manuscript. we consider it a minor limitation to the study. The effects of stress and administration of anesthetic and analgesic Author details drugs are also assumed to be similar for both groups, but Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences, Oslo, Nor‑ it cannot be excluded that the drugs have masked the sur- way. Department of Veterinary Clinical Sciences, Faculty of Health and Medi‑ gical stress response and hence masked a potential small 3 cal Sciences, University of Copenhagen, Copenhagen, Denmark. Department difference between groups. A previous study has shown of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands. Department of Production higher CRP concentrations after canine OHE performed Animal Clinical Sciences, Faculty of Veterinary Medicine and Biosciences, by inexperienced surgeons [27]; however, since we used 5 Norwegian University of Life Sciences, Oslo, Norway. Hill’s Pet Nutrition Inc, the same, experienced surgeon for all procedures, this Topeka, KS, USA. is not relevant for the current study. The dogs were only Acknowledgements followed for 24 h, and a follow-up to assess wound heal- The authors wish to thank Professor Thomas Eriksen for valuable input during ing or inflammatory complications was not carried out. data collection, analysis, and manuscript preparation. In humans, increased perioperative concentrations of Competing interests glucose and CRP have been described as risk factors for The authors declare that they have no competing interests. postoperative infections [19, 52]. A study with longer Availability of data and materials follow-up of the animals with regards to complications The datasets used and analyzed during the current study are available from resulting from surgery would have been of value. the corresponding author on reasonable request. Moldal et al. 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Acta Veterinaria ScandinavicaSpringer Journals

Published: May 30, 2018

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