C-reactive Protein as a Quantitative Biomarker in Female Dogs Undergoing Laparoscopic and Open Elective Ovariectomy

Injury or inflammation to the animal body resulted in the initiation of the acute-phase response (APR). Surgical maneuvering involves removal of a substantial fraction of the diseased tissue that sequence to the inflammation. Many plasma proteins increase in concentration in response to inflammatory stimuli, but C-reactive protein is considered as the most suitable markers due to their rapid and substantial increase in concentration and short half-lives, thus decreasing rapidly when the stimulus ceases (Whicher and Dieppe, 1985). An APR to surgery has been noted and an indication that the changes in APR protein follow a specific pattern with respect to time has also been reported (Aronsen et al., 1972 and Wandall, 1974).
       
The acute phase protein (CRP) is a plasma protein, concentration of which increases in response to tissue damage (Pepys, 1981). Colley et al., (1983) explored an increase in plasma concentration of CRP during surgery, which reach on peak at 48 hours. They suggested that after surgery there is a maximal capacity for production of the acute phase proteins and that this capacity is reached with only a limited degree of trauma. A contrasting view has been argued by Dominioni et al., (1980) who suggested that there was a close correlation between increases in CRP concentration and the magnitude of operation. Rothenburger et al., (1999) investigated the response of C-reactive protein in cardiac surgery. CRP levels were significantly elevated as compared to baseline on all postoperative days investigated. High CRP indicates either acute phase response or local wound problems. Moldal et al., (2018) reported that OHE and OVE induce a surgical stress response with postoperative increases in glucose concentration and CRP, but not signifcant diference between the OHE and OVE.
       
Dabrowski et al., (2009) mentioned that the difficulties in postoperative wound healing were induced by infection with Escherichia coli and Staphylococcus spp., leading to re-increased levels of CRP and SAA immediately after surgery and persistently higher levels throughout the experiment. These findings also indicate that acute phase proteins in bitches undergoing surgery because of pyometra are useful markers for monitoring the postoperative period. The CRP response varied according to the degree of surgical trauma on 3-standardized levels indicating CRP as an inflammatory activity indicator and monitoring markers. Hirvonen and Pyorala (1988) opined that the acute phase protein as an objective marker of the severity of inflammatory processes enable early detection of complications in patients undergoing surgery and prompt institution of decreased therapy. CRP is used as a diagnostic marker for detection of systemic inflammation in dogs (Eckersall and Schmidt, 2014 and Nakamura et al., 2008) and quantitative measurements of canine CRP concentrations have been possible for more than 2 decades (Yamamoto et al., 1993). CRP, a useful biomarker of infection, is produced exclusively by the liver. The aim of this study was to assess CRP concentrations in female dogs undergoing laparoscopic and open elective ovariectomy.

The studies were conducted on twenty clinically healthy, adult, female dogs brought for elective spaying. The animals were scanned with ultrasonography for abnormalities in the reproductive system. The animals diagnosed for any abnormalities in the reproductive tract were not included in this study. Twenty healthy bitches were randomly assigned into four groups (A, B, C and D) consisting of five animals in each group. The studies were conducted in two phases (I and II). In phase I ovariectomy had been performed in animals of groups A and B with laparoscopy whereas, in phase II ovariectomy in animals of groups C and D were done by open laparotomy incision as per designed below.
 
Experimental design.
 


Instrumentation
 
Standard laparoscopic equipment and instruments (M/s Karlstorz, Germany) were used for this study.
 
Preoperative preparation

Each animal underwent surgery early in the morning. All animals were kept off feed and withheld water for 12 and 6 hours respectively prior to surgery.
 
Preparation of surgical site

The mid-ventral and right lateral flank skin over abdomen were shaved, cleaned and scrubbed with chlorhexidine, wiped with 70% alcohol and painted with povidone iodine.

Anaesthesia

All animals were pre-medicated with Glycopyrrolate at the dose rate of 0.02 mg kg^-1 body wt. I.M. followed by inj. Xylazine HCI @ 1.0 mg kg^-1 body wt I.M after fifteen (15) min later to reduce the general anaesthetic agent, that lead to improved cardiovascular stability and contributes to the provision of balanced anesthesia (Kumar et al., 2020). After 10 minutes later induction had been done with propofol @4mg/kg body wt. and maintained on 2% isoflurane volatile anesthetics.

Surgical procedure

After proper anaesthesia the animals were placed on dorsal recumbency on V-Top table to facilitate cranial displacement of the visceral contents. Four quarter drapes were placed approximately 2 cm lateral to each row of mammary gland, at the xiphoid and the pubis.
 
Phase I

Groups A and B: laparoscopic ovariectomy by bipolar electrocoagulation of ovarian vessels

The capnoperitoneum was established by giving a small skin incision of about 0.5 cm caudal to the umbilicus. By grasping and lifting skin and muscle around the incision site with one hand simultaneously the Veress needle inserted at incision site with other hand directing the tip of the needle caudally. Intra-peritoneal placement of Veress needle was confirmed by injecting 5 ml of normal saline through the needle. The solution should flow without resistance and would not return when trying to aspirate. Carbon dioxide insufflation at a rate of 2 liters/min to creates pressure gradient (10 to 12 mm of Hg) were made after connecting the CO₂ gas hose from the endoflator to the luer-lock of Veress needle and then moderate pneumoperitoneum was established. Respiration and capillary perfusion were closely monitored to check over distension. After attaining a sufficient pneumoperitoneum, Veress needle was removed and a 10 mm safety trocar and cannula was inserted into the peritoneum through the Veress needle insertion site. The trocar was then removed and the gas hose from Veress needle was attached to the cannula. A rigid type 10 mm telescope connected with the light source and digital camera was then introduced through the cannula. Intraperitoneal organs were then visualized thoroughly.
       
All animals of groups A and B were followed the same procedures of pneumo-peritoneum. The telescope placed through the 10 mm port was used to identify the epigastric blood vessels in order to facilitate placement of the two para-median instrument ports (Left and Right side) under camera supervision (Triagular) in group A, These ports were created 1 cm lateral to the mammary teat in the caudal abdomen, being sure to avoid the caudal superficial epigastric artery. These were created by 5 mm threaded cannula and trocar through 1cm incision over skin of the respective site whereas, it was in straight line on lineaalba in group B.
       
All animals in groups A and B were subjected to laparoscopic ovariectomy by electrocautary where the ovarian vessels cauterize by using bipolar laparoscopic forcep. After achieving sufficient capnoperitoneum and trocar placement in the triangular fashion in Group A, the right ovary was grasped by grasping forceps, inserted through right paramedian port. Bipolar laparoscopic forceps was inserted through left paramedian port and cauterization and coagulation of ovarian blood vessels was done by applying bipolar electrocautery and transection of ovarian ligament was done to remove the ovary. The procedure was repeated in reverse order for left ovary. The completely resected ovarian portions were taken outside through caudal port under the guidance of 10 mm laparoscope.
       
Each animal of groups A and B were assiduously inspected in all resected sites for haemorrhage after completion of the procedure. The telescope was taken out and the camera was then detached. Intra-abdominal carbon dioxide gas was allowed to escape through the cannula and evacuation was facilitated by both side abdominal wall compressions. The subcutaneous tissue and muscle of the10 mm port was opposed with one simple interrupted cruciate suture using 1-0 Polyglycolic acid (vicryl), followed by simple interrupted skin suture with fine nylon. Similarly paramedian and lineaalba 5 mm port were also sutured with cruciate suture pattern with PGA and skin closured with nylon in routine manner.
 
Phase II
 
Groups C and D: Ovariectomy by laparotomy
 
For the ovariectomy procedure each dog placed on dorsal recumbancy and four quarter drapes were applied to the surgical field. A ventral midline incision was made in group D whereas, in right flank posterior to the last rib in group C. After performing laparotomy, the ovarian pedicles were clamped using forceps, double ligated by using 1-0 polyglycolic acid (Vicryl) and transected. The proper ovarian ligament was then ligated with suture and the ovaries were removed. The abdominal, subcutaneous tissue and muscle were sutured with polyglycolic acid 1-0 (Vicryl) in cruciate pattern and skin sutured with the nylon routinely. All animals were dressed regularly at the portal site with povidone iodine and antiseptic cream. Broad spectrum antibiotic Amoxycillin and sulbactum @ 10 mg/ kg body wt. (Amoxyrum fort, Virbac Animal Health care P. Ltd.) was given to all animals for five days and analgesic inj. Meloxicam @ 0.3 mg/ kg body wt (Melonex, IntasPharma) was given for three days post surgery.
 
Observations

Plasma samples were used for estimation of C-reactive protein.
 
Post-operative observation
 
General behaviour including discomfort and uneasiness, feeding habit, defaecation, urination, licking of the suture line were observed on post operative days. Each animal was monitored carefully for any other post-surgical complications like emphysema, incision site herniation, wound infection, stitch abscess, etc.
Statistical analysis

Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) were used to compare the means at different time intervals amongst the groups (Snedecor and Cochran, 1994)¹⁶, by using SPSS (2016) computer package.

The Mean±S.E. values for the animals of different groups at various time intervals are given in Table 1 and Fig 1. The pre-treatment CRP values did not show any significant change whereas post treatment values of group B (2.90±0.44) were significantly lower as compared to groups D (6.31±0.79) and C (5.35±0.99). On day 4^th, group D (26.67±2.15) values were significantly higher than groups C, B and A. Group Cvalues were also significantly higher than group B. The highest value in group D might be due to more surgical manipulations and post-operative infection at surgical site in the animals under this group.
 

 

 
The present finding was in accordance with the earlier studies in which a significant increase in CPR was observed on day 4^th post surgical treatment of pyometra in complicated cases in dogs (Dabrowsky et al., 2009). Kanno et al., (2019) also reported a characteristic pattern of c-CRP changes after orthopaedic surgery. The highest CRP observed on Day 4 is likely to result from  the inflammatory  process  and effects of LPS, which activates  the macrophages and release of IL-1 and Il-6-the main inducers of increased biosynthesis of acute-phase protein (APPs) during postoperative period in the liver of dog (Buttenschoen et al., 2001). Pro-inflammatory cytokines, especially IL-6, are actively involved in proper wound healing (Helmy et al., 1999). Excessive secretion of Il-6caused by, for example, infections may lead to difficult wound healing (Ertel et al., 1990) by delaying the apoptosis of polymorphonuclear (PMN) cells (Biffl et al., 1996).
       
OVH has risk of hemorrhage, stump granuloma, stump pyometra, ovarian remnant syndrome and vaginal bleeding these complications are less in OVE (Pearson, 1973 and Wallace, 1991). As OVE  results  in  a smaller incision, better viewing of the ovarian pedicle, complications such as  bleeding, incisional swelling, seroma, infection, dehiscence, delayed healing, self-inflicted trauma and pain are expected to be less (Van Goethem et al., 2006 and DeTora and McCarthy, 2011). Based on the report of short and long term complications in OVH, the present study was planned to perform OVE as elective surgery. The OVE site for open surgical procedures are either through linea alba or left or right flank to remove the ovary. Laparoscopic OVE is also becoming popular because of decrease postoperative stress, shorter recovery periods, decreased hospitalization, improved cosmesis and improved visualization of abdominal organs (Gyr et al., 2001). The acute phase response is a non-specific reaction to any tissue stimulation disturbing the homeostasis e.g., surgery, trauma, infection, or neoplasia and plays an important role as part of the innate immune system (Ceron et al., 2005; Eckersall and Bell, 2010 and Cray et al., 2009). During the acute phase-response concentrations of acute phase proteins will change, reflecting the presence of circulating cytokines (Petersen et al., 2004). In dogs, C-reactive protein (CRP) is a major acute phase protein showing significant increases in concentration as a result of systemic inflammation. CRP is a sensitive and specific marker for systemic inflammatory activity (Kjelgaard-Hansen and Jacob, 2011). The application of postoperative analgesia like administration of NSAID may influence the changes in serum CRP; however, CRP changes in systemic inflammation are not significantly influenced by NSAIDs (Hulton et al., 1985). This makes sense as CRP production is mainly regulated by pro-inflammatory cytokines. It has been reported that canine CRP could serve as a quantitative marker of systemic inflammation, as the observed serum concentrations have been reported to reflect the severity of an inflammation (Caspi et al., 1987). CRP changes in systemic inflammation are not significantly influenced by the use of NSAIDs. This makes sense as CRP production is mainly regulated by pro-inflammatory cytokines and NSAID only modulates prostaglandin synthesis (Hulton et al., 1985).

In laparoscopic ovariectomy release of CRP is significantly lower in comparison to open elective ovariectomy; as tissue manipulation and chance of post-operative infection is significantly lower during laparoscopic ovariectomy in comparison to open elective ovariectomy.