Retrospective studies have shown that diabetes, both type 1 and type 2, is an independent risk factor for postoperative wound infection after cardiac surgical procedures,1–4 with infection rates 2 to 5 times higher in diabetic than in the nondiabetic population.2 A recent retrospective study demonstrated that general surgery patients with hyperglycemia show a 2-fold higher risk of infection.5 No prospective clinical trials have been performed testing whether the duration and degree of glycemic control reduce infection in diabetic patients undergoing surgery. Among diabetic patients, 10% have type 1 and 90% type 2.6 For the type 1 patients, 25% of the adult population have persistent poor glycemic control,7 and in a paper from Scotland, 37% had very poor glycemic control (HbA1c ≥9%).8 Therefore, a large number of type 1 diabetic patients may have long-standing hyperglycemia when presenting for surgery.
Although the benefits of glycemic control in reducing postsurgical infections after surgery have been shown, the serious risk of hypoglycemia associated with perioperative glycemic control has raised concerns, and thus, its use remains a controversial issue.4,5,9,10 The main hypothesis of our preclinical study in type 1 diabetic rats was that glycemic control initiated just before surgery is effective in reducing surgical site infection. The primary aim was to demonstrate that a short-term glycemic control regimen in which insulin treatment is initiated just hours before surgery reduces postoperative infection compared with no glycemic control. The secondary aim was to show that short-term glycemic control is as effective as long-term glycemic control, in which insulin treatment is initiated 2 weeks before surgery, in reducing surgical site infection.
Diabetic Rat Model
All experimental procedures in this study were approved by the Animal Care and Use Committee of Rush University Medical Center and conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council). To induce diabetes, male Sprague-Dawley rats (Sasco, Charles River, Wilmington, MA; 160–180 g) were sedated with 1.2% isoflurane in oxygen and injected with a single dose of 50 mg/kg streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO) to destroy pancreatic β-cells.11 The drug was freshly prepared in 0.05 M, pH 4.0 citrate buffer and injected percutaneously into the lateral tail vein in a volume of 0.2 mL, as previously done by our group.12,13 Six days after STZ injection, blood glucose levels were tested with a commercial meter and test strips (FreeStyle; Abbott, Alameda, CA) from a small drop of blood obtained by tail prick (27G needle). Animals with blood glucose levels >250 mg/dL (14 mM) were considered diabetic. The nondiabetic group had the citrate buffer vehicle injected into the lateral tail vein. Although this is a type 1 diabetic model, STZ diabetic rats can survive more than a year without any treatment.14
Insulin Treatment for Glycemic Control
The study had 4 treatment groups (Fig. 1), all with surgery and bacterial injection on the same day:
- Short-term group: Diabetic rats with insulin treatment started just before (short-term glycemic control) surgery and infection exposure and continued until the end of study.
- Long-term group: Diabetic rats with insulin treatment started 2 weeks before (long-term glycemic control) surgery and infection exposure and continued until the end of study.
- Untreated diabetic group: Diabetic rats with no insulin treatment (no glycemic control).
- Nondiabetic rats
All rats had blood glucose levels measured 21 days after STZ or vehicle injection to verify that diabetes was still present in STZ-injected rats. Diabetic rats were randomized into 3 groups: a short-term insulin-treated group, a long-term insulin-treated group, and an untreated diabetic group. Nondiabetic rats served as a comparison group.
The long-term group received insulin treatment starting day 21 after 50 mg/kg STZ injection (Fig. 1). These rats were sedated with 1.2% isoflurane in oxygen, and the abdominal skin shaved and swabbed with antiseptic solution. A skin puncture was made over the upper abdomen region with a 16G needle, the needle withdrawn, and a 14G trocar then introduced into the subcutaneous space. One whole (7-mm long) and one-half insulin slow-release capsules (Linplant; LinShin, Toronto, Canada) were then delivered through the trocar with a stylet until it was clearly seen in the subcutaneous space. The puncture hole was closed with tissue adhesive (Vetbond, 3M, St. Paul, MN). Blood glucose was retested after 6 days (on day 27 after STZ injection) to confirm glycemic control. The 1½ insulin capsules deliver 3 insulin units per day (U/day), as previously described.11,13 This insulin dose protocol had been shown to reduce blood glucose to within normal limits in STZ diabetic rats and over a 2-week period partially reverses neurological deficits in the STZ model.11
At 34 days after STZ injection, the untreated STZ-induced diabetic rats were retested to ensure that hyperglycemia (>250 mg/dL) was still present in these animals. These untreated diabetic rats were then randomized on day 35 into the short-term insulin-treated and untreated diabetic rat groups (Fig. 1). Early in the morning of surgery (day 35 after STZ), the short-term group received a bolus subcutaneous injection of 6U of intermediate acting NPH human insulin (Novo Nordisk, Bagsvaerd, Denmark), as previously described.11,13 Then, just before surgery (2–3 hours after the insulin bolus), the short-term group had insulin slow-release capsules (3 U/d) implanted subcutaneously (Fig. 1) using the same method that the long-term group had on day 21.
Then, all 4 groups of rats (short-term treated diabetic, long-term treated diabetic, untreated diabetic, and nondiabetic) underwent the same thigh surgery and bacteria injection described in the surgical infection model section below. Blinding of groups was not possible due to the polyuria (including wet cage bedding) in the short-term treated diabetic and untreated diabetic rats. The short-term and long-term groups continued to receive glycemic control until euthanized.
Surgical Infection Model
The bacterial species chosen to induce a muscle surgical infection was Staphylococcus aureus (S aureus; American Type Culture Collection, No. 29213), which is the most common cause of surgical wound infections.15,16 An important feature of this bacterium is the formation of abscesses at the inoculation site, which makes it convenient for quantifying bacterial burden in tissue. Cultures were propagated in tryptic soy broth. The day before surgery and bacterial injection, frozen stock from the initial propagation of bacteria was grown overnight (37°C incubator). Bacterial titers were determined as colony forming units (CFUs) by serial dilution and plating, as previously described,17,18 to provide a 5 × 108 CFU/mL concentration (based on optical density) of S aureus for the morning of surgery.
For surgery, rats were anesthetized with 1.5% isoflurane in oxygen provided via a nose cone. The surgery was performed with sterile instruments, sterile surgical gloves, and aseptic techniques as follows. The left thigh was shaved and disinfected with alcohol swabs (3 times) followed by a topical antiseptic solution (chlorhexidine gluconate 4%) applied to the skin. A 2-cm-long skin incision was made with a No. 15 scalpel blade to expose the biceps femoris muscle. The skin margins were retracted, and a 7-0 polypropylene suture was placed on the surface of the muscle for later identification of the injection site. Using a 25G needle and 1 mL syringe, 1 × 108 CFU of S aureus were slowly injected into the biceps femoris muscle in 0.2 mL volume,19 adjacent to the 7-0 suture. A plastic tubing sheath over most of the needle limited the injection depth to 2 mm. At the end of surgery, all skin margins were closed with 4-0 nylon sutures. The topical antiseptic solution was again applied to the skin, and the animal recovered from anesthesia.
Postoperative Infection: Bacterial Muscle Burden
At 3 or 6 days after surgery and S aureus muscle injection, blood glucose was measured in all rats to confirm glycemic control in the long-term and short-term groups. Rats were then euthanized with carbon dioxide in a closed chamber. Under aseptic conditions, the left biceps femoris muscle was exposed, and a section selected around the 7-0 suture (that had been implanted before bacterial injection). A 10 × 5 mm area of infected muscle, 5-mm thick, was then removed from the body and used for determination of bacterial loads as follows. The muscle was weighed (typical value = 0.25 g), minced with a razor blade, and homogenized (PowerGen 125, Fisher Scientific, Pittsburgh, PA; 7 mm probe, at full speed for 10 sec × 3 times) in 1 mL of sterile phosphate-buffered saline. The bacterial loads were determined by serial dilution and plating, as previously described.17,20 Briefly, the homogenized muscle mixture underwent 10-fold serial dilution in phosphate-buffered saline to produce 10−1 to 10−5 dilutions in 1 mL volume, plus an undiluted sample 100. One hundred-microliter aliquots of each muscle solution (10−5–100) were plated on tryptic soy agar plates and incubated for 24 hours at 37°C. The next day, muscle tissue bacterial counts (CFU) were determined from plates with 30 to 300 colonies. The final bacterial burden is expressed as CFU per tissue wet weight. The 3- and 6-day time points were chosen to represent the acute and early chronic phases of S aureus infection in rat muscle.21
Postoperative Infection: Systemic Measures
White blood cell (WBC) count expressed as K/μL was obtained from tail vein blood samples (typically 200 μL) just before surgery and just before being euthanized on day 3 or day 6 after infection exposure. After dilating the veins by dipping the tail in a 40°C water bath, venipuncture was performed with a 25G butterfly needle with tubing cut to 3 cm length. While warming the tail may influence the baseline WBC count, the same technique was used for all groups in the study. Samples were collected in small K2-EDTA tubes (Microtainers, BD, Franklin Lakes, NJ) and analyzed within an hour after collection using a Cell-Dyn 1700 hematology analyzer (Abbott Diagnostics, Abbott Park, IL). Bacteremia was assessed on the day the rats were euthanized (day 3 or day 6 after surgery) by adding 10 μL of the tail vein blood sample to 90 μL sterile saline.22,23 The 100-μL aliquot was analyzed for S aureus bacteria counts by plating on tryptic soy agar plates. The blood bacteria level is expressed as CFU/mL blood. Body temperature was measured with a lubricated flexible rectal probe (Yellow Springs, model 402, Yellow Springs, OH) in gently restrained rats, just before surgery and just before being euthanized on day 3 or day 6 after infection exposure.
Power analysis was based on the primary outcome: difference in bacterial muscle burden between the short-term group and the untreated diabetic rats at 6 days after surgery and S aureus muscle injection. A study with S aureus hindpaw injection in type 1 diabetic mice versus nondiabetic mice demonstrated that at 7 days after infection, the mean CFU/g tissue was 3 log orders lower in the nondiabetic mice.24 If we hypothesized that in our study the short-term group CFU/g tissue would also be similarly reduced at 6 days compared with untreated diabetic rats, then for 80% power, α = 0.05 in the 2-tailed t test for means, and an effect size = 1.4, the required n per group = 9.25
As secondary outcomes, differences among the 4 treatment groups at each time point were analyzed using an analysis of variance structure in a general linear model with Tukey adjusted post hoc analysis. All analyses were performed using statistical software (SAS version 9.2, Cary, NC). Changes in group measures between time points were analyzed using a paired t test and Holm-Bonferroni adjustment for multiple comparisons. Due to the highly skewed nature of the blood CFU/mL distribution (mode = 0), this secondary measure was dichotomized and analyzed as an incidence using first an omnibus test of overall difference among incidence for the 4 groups and then if significant a Fisher exact test of each group versus the 3 others collapsed into 1 category (2 × 2 matrix). The omnibus test was conducted using Monte Carlo estimation of exact test based on the likelihood ratio estimate. Distributional assumptions were evaluated (i.e., Kolmogorov-Smirnov D test, normal Q-Q plots) and if deemed to be violated, corrective measures were taken (i.e., log transform to normalize distributions). To test model assumptions that residuals are normally distributed, linearly related, and independent with constant variance, we examined residual plots. To evaluate independence, constant variance (homogeneity of variance), and nonlinearity of the regression function, we examined plots of residuals versus fitted (predicted) values, Studentized residuals versus fitted values, and box plots. To evaluate normality, we examined Q-Q plots of residuals and distribution plots of residuals with normal fit lines.
Specifically, for models representing the significance tests in tables showing changes in body temperature and WBC, there were no significant departures from normality for these models and resulting contrasts, and examination of the residuals did not identify violations of assumptions of homogeneity of variance, normality, or linearity. Although some models had mild heterogeneity issues, none of these was severe.
For the bacterial burden in muscle outcomes, distributional analysis indicated that the moderate skewness identified may be corrected using transformations. We made conservative choices on the use of transformations, so that the bacterial burden in muscle variables was all log (base 10) transformed. A sensitivity analysis of log versus non-log-transformed data showed no change in significance for any outcome, but several log-transformed data resulted in less highly significant P values (higher P values). The more conservative log-transformed data are presented.
To provide support for situations where “no change” or “no difference” was indicated, we produced figures presenting the confidence intervals for the differences described. All these differences were compared with the nondiabetic control so that the Dunnett post hoc test adjustment was used to control familywise error rate. As can be seen on the plots (Fig. 2, C and D), the 95% confidence intervals are sufficiently narrow and are not significantly different from 0 (i.e., not significantly different).
Achievement of Glycemic Control
By day 6 after STZ injection, rats had higher blood glucose levels than the vehicle-injected nondiabetic animals (Fig. 2, A–D). On the day before surgery (day 34), the long-term group no longer had elevated blood glucose levels (Fig. 2, A–D). On the day the rats were euthanized, at 3 or 6 days of infection, neither the long-term group nor the short-term group had elevated blood glucose levels (Fig. 2, A–D). Collectively, these data indicated that STZ injection resulted in hyperglycemia and that glycemic control using long-term or short-term protocols were effective in controlling blood glucose levels throughout the infection periods (3 days or 6 days). Also of note, hypoglycemia (blood glucose <40 mg/dL) did not occur in the long-term or short-term groups (lowest value = 50 mg/dL), although with once-a-day glucose monitoring we may have missed transient instances of hypoglycemia.
Short-Term and Long-Term Glycemic Control Treatments Reduce Bacterial Infection
When all 4 treatment groups were compared, the bacterial burden in the biceps femoris muscle in rats euthanized 3 days after surgery (Fig. 3A) was significantly higher in untreated diabetic rats (~2 log orders) than that in nondiabetic animals or both groups of insulin-treated diabetic rats (all P < 0.003). There was no statistical difference among the short-term, long-term, or nondiabetic rats at 3 days of infection (all P > 0.98). The bacterial burden in muscle in rats euthanized 6 days after surgery was very high in untreated diabetic rats (Fig. 3B) but was reduced greatly by nearly 3 log orders in the nondiabetic, and the long-term and short-term groups (all P < 0.0001). There was no statistical difference among the short-term, long-term, and nondiabetic rats at 6 days of infection (all P > 0.99).
No Bacteria in Blood of Short-Term and Long-Term Glycemic Control Groups
In rats euthanized 3 days after surgery, S aureus was not detected in blood samples of any of the nondiabetic, long-term treated, or short-term treated rats, but 1 of the 6 untreated diabetic rats exhibited S aureus in blood (P = 0.9999). For rats euthanized 6 days after surgery, S aureus was not detected in blood samples of any of the nondiabetic, long-term, or short-term rats but was detected in some of the untreated diabetic rats. The overall incidence of bacteremia were different among the 4 groups (omnibus test of difference P = 0.0471) highlighted by the incidence in the untreated diabetic rats where 3 of 9 had bacteremia compared with zero incidence of 26 in the rest of the groups combined (P = 0.0143). The actual blood bacteria values of those untreated diabetic rats were 1000, 2700, and 2800 CFU/mL.
Body Temperature and WBC Counts
Body temperature just before surgery, in rats euthanized 3 days after surgery, was lower in the remaining untreated diabetic rats (short-term and untreated groups) compared with that in the nondiabetic rats (Table 1). After 3 days of infection, neither was there any difference in body temperature among groups (all P > 0.18) nor was there any increase from the value before surgery for any group (all P > 0.18). For rats euthanized 6 days after surgery, the results for body temperature just before surgery were again lower in the remaining untreated diabetic rats (short-term and untreated groups) compared with those in the nondiabetic rats (Table 1). After 6 days of infection, body temperature was lower in the untreated diabetic group than that in the nondiabetic group (Table 1). At the 6-day time point, there were no significant increases from the value before surgery for any group (all P > 0.46).
For rats euthanized 3 days after surgery, the WBC count just before surgery was lower in the remaining untreated diabetic rats (short-term and untreated groups) than that in the nondiabetic rats (Table 2). After 3 days of infection, although WBC increased numerically in all 4 treatment groups from the level before surgery, the increase was only significant for the short-term group (P = 0.0005; 76% increase). At the 3-day time point, there were no statistical differences among the 4 groups (all P > 0.05). After 6 days of infection, although WBC increased numerically in all 4 treatment groups from the level before surgery, the increase was not significant (Table 2). At the 6-day time point, there were no statistical differences among the 4 groups (all P > 0.55).
Patients presenting for surgery with elevated blood glucose levels on the day of surgery pose a dilemma for the perioperative physician. The anesthesia and surgical team can (1) proceed with the surgery as planned; (2) postpone the surgery and have the patient placed on a glycemic control protocol; or (3) implement glycemic control and proceed with surgery. Currently, there are no standard guidelines addressing this particular clinical dilemma for noncardiac surgery patients. However, there are overall recommendations on perioperative glycemic control in noncardiac surgery patients.5,26,27 These recommendations are fairly similar to those for cardiac surgery patients,28 and typically use a cutoff for maintaining blood glucose <180 mg/dL, to reduce adverse outcomes including infection. Using a well-established model for hyperglycemia (STZ-induced diabetes), which has been used for almost 50 years as a model for type 1 diabetes,11–14 and exposure to S aureus (a relevant pathogenic bacteria in such settings),15,16 we evaluated the efficacy of short-term glycemic control (just before surgery) and long-term glycemic control (2 weeks before surgery) in reducing bacterial infection in diabetic animals undergoing surgery. We found that STZ-induced diabetes renders these rats highly susceptible to thigh muscle infection secondary to S aureus (which we used as a general surgery model for infection). Our data are consistent with previous reports that demonstrated enhanced susceptibility of type 1 (nonobese diabetic mice) and type 2 (db/db) diabetic mice to bacterial infection.24,29 In these mouse diabetic models and our STZ model, the blood glucose levels are atypically high. Nonetheless, clinical studies would suggest that reducing perioperative glucose levels should reduce the development of infection.30
Importantly, our data demonstrate for the first time in a diabetic animal model that a short-term glycemic control regimen initiated just before surgery and infection exposure is effective in reducing surgical site infection and, moreover, is just as effective in reducing surgical site infection as a long-term glycemic control that began 2 weeks before surgery.
S aureus did not appear in the blood samples of any of the nondiabetic rats nor the long-term or short-term treated diabetic rats, at 3 or 6 days after an intramuscular injection of 1 × 108 CFU bacteria. However, in bacteria-injected diabetic rats with no insulin treatment, S aureus appeared at 6 days in the blood samples of some of the rats, indicating that short-term (and long-term) insulin treatment is effective in reducing hematogenous spread of infection.
Our finding of decreased body temperature in untreated STZ diabetic rats before surgery compared with nondiabetic rats is consistent with previous studies on STZ rats.31,32 Rodents have a decreased systemic inflammatory response to bacterial loads33 and that may account for the lack of an increase in body temperature (i.e., fever) in our model on day 3 or day 6 after surgery and infection exposure. WBC count was lower in untreated diabetic rats than that in nondiabetic rats, similar to the observation in another study.34 At 3 days after surgery and infection exposure, but not at day 6, only the short-term insulin-treated group showed a significant and large increase in WBC count. Whether this increased WBC count at the start of glycemic control was critical to these diabetic rats clearing the bacterial muscle burden is unclear.
The factors by which hyperglycemia makes diabetic patients more susceptible to infection are varied.2–4,7,35–38 Reduced neutrophil activity in diabetic patients35 and patients with acute hyperglycemia36 has been proposed as a mechanism that increases the risk of infection. In diabetic patients, both chemotaxis and the oxidative potential of polymorphonuclear leukocytes are reduced.35 Decreased complement function and increased inflammatory cytokine levels have also been implicated as contributing to the immune dysfunction of hyperglycemia.36 The mechanism by which glycemic control allows diabetic animals to limit microbial growth at a surgical site, similar to nondiabetic animals, is unclear. Type 1 diabetic mice (nonobese diabetic) have decreased respiratory burst (oxidative potential) in neutrophils from whole blood compared with nondiabetic animals,24 but it is not known if glycemic control can restore this activity. Type 2 db/db diabetic mice have decreased superoxide production from blood neutrophils after a 7-day S aureus infection, and this is restored in diabetic mice that received twice a day NPH insulin injections for 7 days before bacteria injection and continued for 7 days after.39 However, it is not clear if blood neutrophil oxidative activity alone can account for the antibacterial neutrophil activity in the tissue.
While the STZ-injected rat is a widely used and accepted model of hyperglycemia,11–14 we recognize that our results on glycemic control reducing postoperative infection cannot be directly extrapolated to diabetic patients. Type 1 diabetes is a complex, multifactorial disease, and the STZ model only represents a few aspects of the disease, namely insulinopenia and hyperglycemia.40 However, retrospective analyses in patients undergoing noncardiac surgery have shown the postoperative blood glucose level, not the preoperative blood glucose level, to be the most important predictor of surgical site infection.41,42 Therefore, our finding that short-term glycemic control initiated just before surgery and infection exposure in animals with established diabetes effectively reduces surgical site infection suggests that glycemic control begun as late as the day of surgery may be adequate. While in humans evidence is mounting that reduced infection with insulin treatment is due to the lower blood glucose level, rather than the insulin itself,4 it cannot be excluded that in the animal experiments there is also a direct effect of insulin in clearing bacterial burden.
Although not intended in the study, the slow-release insulin capsules used in our study put the animals in the low normoglycemic range; so in effect we had tight glucose control. Tight perioperative glycemic control in patients (intensive insulin therapy) has an increased incidence of hypoglycemia, which is not a benign condition.4,10,27,43,44 In addition, the consequences of severe hypoglycemia due to intensive insulin therapy can differ among the surgery patient population,44 being of most concern for brain injury patients.27 Future animal experiments should be designed with blood glucose levels maintained over a higher range (e.g., 110–180 mg/dL). It should be noted that in addition to diabetic hyperglycemia some patients may have stress-induced hyperglycemia.27,44,45 Major surgery leads to metabolic stress with transient hyperglycemia even in nondiabetic patients.26
In conclusion, our study in type 1 diabetic rats has shown that even short-term perioperative glycemic control reduces surgical site infection. Well-designed clinical studies will be necessary to validate that patients presenting for various types of surgery in a diabetic state can undergo surgery with a decreased risk of postoperative infection if glycemic control can be instituted just before surgery commences.
Name: Jeffrey S. Kroin, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Jeffrey S. Kroin has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Asokumar Buvanendran, MD.
Contribution: This author helped design the study, conduct the study, and write the manuscript.
Attestation: Asokumar Buvanendran has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Jinyuan Li, MD, PhD.
Contribution: This author helped conduct the study.
Attestation: Jinyuan Li has seen the original study data and approved the final manuscript.
Name: Mario Moric, MS.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Mario Moric has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Hee-Jeong Im, PhD.
Contribution: This author helped design and conduct the study.
Attestation: Hee-Jeong Im reviewed the analysis of the data and approved the final manuscript.
Name: Kenneth J. Tuman, MD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Kenneth J. Tuman reviewed the analysis of the data and approved the final manuscript.
Name: Sasha H. Shafikhani, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Sasha H. Shafikhani has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Avery Tung, MD.
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