Impact of the duration of preoperative glycemic control on perioperative neutrophil functions in diabetic mice with chronic hyperglycemia: a laboratory research report

BACKGROUND: The risk of surgical site infection has been reported to be higher in patients with poorly controlled diabetes. Since chronic hyperglycemia impairs neutrophil functions, preoperative glycemic control may restore neutrophil function. However, long-term insulin therapy may lead to a delay in surgery, which may be a problem especially in cancer surgery. It is therefore unfortunate that there was no study to investigate the optimal duration of perioperative glycemic control for diabetes with chronic hyperglycemia. Therefore, we investigated the effects of preoperative long-term insulin therapy and short-term insulin therapy on perioperative neutrophil functions in diabetic mice with chronic hyperglycemia. METHODS: Five-week-old mice were administered streptozotocin to induce diabetes. We divided the diabetic mice into three groups: a group of mice with untreated diabetes (untreated group), a group of mice that received insulin therapy for 6 hours before surgery (short-term group) and a group of mice that received insulin therapy for 5 days before surgery (long-term group). A group of non-diabetic mice were used as a control group. At 14 weeks of age, abdominal surgery with intestinal manipulation was performed in all four groups. We carried out a phagocytosis assay by fluorescent microspheres and a reactive oxygen species (ROS) production assay by DCFH-DA (2’,7’-dichlorodihydrofluorescin diacetate) before and after surgery using FACSVerse™ with BD FACSuite™ software. Phagocytosis rate (%) was calculated by dividing the number of phagocytic beads by neutrophil count. Fluorescence from DCF (dichlorofluorescein) in the neutrophils was measured and expressed as mean fluorescence intensity (MFI). RESULTS: Blood glucose was lowered by insulin therapy in the short-term and long-term groups before the operation. Neutrophilic phagocytosis activities before and after the operation were significantly restored in the long-term group compared with those in the untreated group (before: p = 0.0008, after: p = 0.0005). penicillin, μg/ml sterptomycin, After incubation with purified anti-CD16/CD32 antibody CA, USA) 10 minutes for blocking Fc receptors, cells were incubated with Pacific Blue™ anti-mouse Ly-6G antibody CA, 30 minutes on in the dark, and washed with FACS We analyzed the Ly-6G-positive cells with fluorescence of microspheres by using purified

for 5 days before the operation but not by insulin therapy for 6 hours before the operation.

Background
It has been reported that about 10% of patients undergoing elective surgery have diabetes mellitus (DM) [1,2] and that about 20% of those patients have poor glycemic control preoperatively [2,3].
The risk of postoperative infectious complications was shown to be significantly higher in patients with diabetes than in patients without diabetes [1]. Furthermore, poorly controlled diabetes was reported to be associated with an additional higher risk of surgical site infection (SSI) [4][5][6].
Since chronic hyperglycemia induces impairments of neutrophil functions including phagocytosis, chemotaxis and bactericidal activity [7,8], preoperative glycemic control may restore neutrophil functions in patients with chronic hyperglycemia, which may result in a reduction in the incidence of SSI. Indeed, the Centers for Disease Control and Prevention Guideline recommends lowering perioperative blood glucose levels [9]. However, information on the optimal duration for preoperative glycemic control is not provided in the guidelines.
In the ADVANCE study, it was shown that long-term insulin control is beneficial for patients with type 2 DM [10]. However, preoperative long-term insulin therapy may lead to a delay in surgery, which may be a problem for some types of surgery, especially cancer surgery. It is therefore unfortunate that there is little information on the effect of duration of preoperative glycemic control on perioperative neutrophil functions in patients with diabetes, and there has been no study in which the effects of preoperative short-term insulin therapy and long-term insulin therapy were compared.
We therefore conducted a study to compare the effects of preoperative short-term insulin therapy and long-term insulin therapy on perioperative neutrophil functions in diabetes mice with chronic hyperglycemia.

Methods
This study was approved by the Kobe University Animal Experiment Committee (approved on October 23, 2017, No. P151004). Male C57BL/6J mice (4 weeks old; body weight, 16-18 g) were purchased from Japan SLC (Shizuoka, Japan). The animals were maintained in a temperature-and humiditycontrolled room (22-25℃, 50-60%) on a 12-hour light-dark cycle. They had free access to normal water and normal diet. The study flow is summarized in Figure 1.

Mice with streptozotocin-induced chronic diabetes
We prepared streptozotocin (STZ) (Wako Pure Chemical Industries, Osaka, Japan) dissolved in sodium citrate buffer (pH 4.5). At the age of 5 weeks, mice were intraperitoneally administrated STZ (50 mg/kg body weight) for 5 consecutive days to induce diabetes according to prior studies by Frisch et al. and Jackson et al. [11,12]. For non-diabetic controls, mice were administered only the buffer at the age of 5 weeks.
Diabetic mice and non-diabetic mice were fed a normal chow diet for 8 weeks. We measured blood glucose levels in tail vein blood using a glucometer (Glutest Neo alfa®, Sanwa Kagaku Kenkyusho, Japan) every 2 weeks. We defined the development of diabetes as random blood glucose level ≧ 300 mg/dL (17 mM) [13] at the age of 14 weeks. We also measured body weight every 2 weeks.

Insulin therapy
We divided the diabetic mice into three groups (Figure 1). Neutral protamine Hagedorn (NPH) insulin (Humulin N; Eli Lilly, Indiana, USA) was injected subcutaneously to maintain the blood glucose level below 200 mg/dL using an insulin sliding scale (supplemental file 1). Insulin was injected at 6 hours before the operation in the short-term groups, and insulin was injected every 12 hours for 5 days before the operation in the long-term group. Determination of the dose of insulin and measurement of blood glucose level were performed before and 6 hours after insulin administration during the period of insulin therapy.
Daily insulin sensitivity factor (ISF) as a surrogate of insulin sensitivity [14], which is the drop in blood glucose caused by 1 unit of insulin, was calculated by using the following formula [15]: ISF ( mg dL -1 U -1 ) = change in blood glucose (mg/dL) / amount of insulin (U). A group without any insulin therapy before the operation was used as a control group.

Surgical procedure
We performed intestinal manipulation in each mouse at 14 weeks of age under general anesthesia with 3.5% sevoflurane and air, as shown Figure 2. Each mouse was placed in the supine position on a heating pad (37℃) during the procedure and shaved the hair (a). After injection of 1% lidocaine (Maruishi Pharmaceutical, Osaka, Japan), a vertical incision of 0.5 cm in length was made in the middle of the abdomen (b-1, b-2). The small bowel luminal contents were moved by using two moist and sterile cotton sticks from the pylorus to the cecum [16] (c). The surgical wound was closed with 5-0 nylon (Natume Seisakusho Co., Tokyo, Japan) (d-1, d-2). After the surgical procedure, EMLA® cream including 2.5% lidocaine and 2.5% prilocaine (Sato Pharmaceutical Co, Tokyo, Japan) was applied to the surgical site for analgesia. The animals were placed under a heating lamp until recovery from anesthesia. After completing the experiment, mice were euthanized by cervical dislocation.

Analysis of neutrophil function
Neutrophil functions were examined before and 24 hours after surgery by using two assays. A neutrophil phagocytosis assay was carried out by using fluorescently labeled microspheres. Briefly, neutrophils were isolated from heparinized peripheral blood by centrifugation after lysis of red blood An assay of reactive oxygen species (ROS) generated by neutrophils was carried out according to a previous study [17]. Heparinized peripheral blood was incubated for 30 minutes at 37 ℃ with 5 μM 2',

Statistical analysis
Data are shown as median values with interquartile range (IQR). A comparison between two groups was performed using the Mann-Whitney U test for unpaired data and Wilcoxon's signed rank test for paired data with Prism 8 (GraphPad Software, San Diego, USA).
Our primary null hypothesis was that insulin treatment may not alter the neutrophil functions of diabetic mice. Therefore, we considered the untreated group as a reference for comparisons among 4 groups. Considering the bias of multiple comparisons (3 times), a p-value < 0.0167 was considered to indicate statistical significance for the analysis of blood glucose levels and result of neutrophil function tests. For comparisons of insulin demand and ISF value, we considered the value on the first day in the long-term group as a reference. For this analysis, a p-value < 0.01 was considered to indicate statistical significance for adjusting the bias of multiple comparisons (5 times).
To calculate the sample size for the current study, we considered on absolute difference of 10% in the phagocytosis rate and 500 in ROS to be meaningful. Assuming standard deviations of 8% for phagocytosis rate and 250 for ROS, an α level of 0.0167 and a power of 0.80, approximately 15 mice and 6 mice were required in each cohort.

Results
Preoperative insulin therapy and blood glucose levels Figure 3 shows the dose of insulin administration each day. On the first day, the median insulin dose in long-term group was 7 U (IQR: 4.5-10), which was not significantly different from the median dose of 6 U (IQR: 5.25-10) in the short-term group (p = 1.00). In the long-term group, the insulin dose required to control glucose levels gradually decreased, and the doses were significantly lower on the 3rd, 4th and 5th days of insulin therapy than on the first day (3rd, 4th, and 5th days vs. first day: p = 0.0001, < 0.0001, and < 0.0001, respectively). Figure 4-A shows preoperative blood glucose levels. In the non-DM group, blood glucose was within the normal range. In the untreated group, blood glucose level was around 600 mg/dL. In the shortterm group, blood glucose level was 600 mg/dL for 5 days and then decreased to within the normal range before the operation. In the long-term group, blood glucose level was less than 200 mg/dL for 5 days before the operation. Figure 4-B shows the blood glucose levels immediately before the operation. The preoperative blood glucose level in the untreated group was significantly higher than the levels in the other three groups.
Insulin sensitivity factors during the insulin therapy period Figure 5 shows the ISFs in the long-term and short-term groups during the insulin therapy period. The median ISF on the first day in the long-term group 51.3 mg dL − 1 U − 1 (IQR: 43.1-76.1), which was not significantly different from the median ISF of 67 mg dL − 1 U − 1 (IQR: 46.7-83.3) in the short-term group (p = 0.47). In the long-term group, ISF gradually increased and was significantly higher on the 3rd, 4th and 5th days of insulin therapy than on the first day (3rd, 4th, and 5th days vs. first day: p = 0.0061, 0.0004, and < 0.0001, respectively).
Phagocytosis activities before and after surgery Figure 6-A shows the phagocytosis rates before surgery in the four groups. In the short-term group, the median phagocytosis rate was 16.2% (IQR: 13.6-25.6), which was not significantly different from the median rate of 19.0% (IQR: 16.8-21.0) in the untreated group (p = 0.87). In the long-term group, the median phagocytosis rate was 25.1% (IQR: 21.5-30.1), which was significantly higher than that in the untreated group (p = 0.0008). The median phagocytosis rate in the long-term group was comparable to the median rate of 27.9% (IQR: 23.3-34.2) in the non-DM group (p = 0.63). Figure 6-B shows the phagocytosis rates after surgery in the four groups. The trends were similar to those before surgery. In the short-term group, the median phagocytosis rate was 11.7% (IQR: 9.8-20.6), which was not significantly different from the median rate of 12.1% (IQR: 8.8-15.1) in the untreated group (p = 0.41). In the long-term group, the median phagocytosis rate was 22.2% (IQR: 18.3-32.0), which was significantly higher than that in the untreated group (p = 0.0005). The median phagocytosis rate in the long-term group was comparable to the median rate of 17.3% (IQR: 11.7-25.1) in the non-DM group (p = 0.14).

Key findings
The aim of the present study using mice with STZ-induced chronic diabetes was to determine the impact of the duration of preoperative insulin therapy on the phagocytosis activity and ROS production activity of neutrophils before and after surgery. We found that impaired phagocytosis activity induced by chronic diabetes was restored to a level similar to that in the non-DM group by preoperative insulin therapy for 5 days but not by preoperative insulin therapy for 6 hours.

Related animal studies
The literature includes two prior relevant studies. Yano et al. conducted a study using 16-week-old diabetic db/db mice and high fat diet-fed mice [18]. Blood glucose levels in their diabetic mice were approximately between 216 and 252 mg/dL. Blood glucose level gradually decreased with insulin treatment and was > 126 mg/dL after 7 days of insulin therapy. Their insulin treatment had significantly improved preoperative phagocytosis activity of neutrophils and decreased the maximal diameter of surgical site infection in both mice. Insulin treatment significantly increased superoxide production in db/db mice but decreased it in high fat diet-fed mice. They did not assess neutrophilic function after the operation.
Kroin et al. conducted a study using Sprague-Dawley rats with diabetes induced by STZ [19]. Blood glucose levels in their rats were over 250 mg/dL. The insulin therapy for 2 weeks achieved a normal blood glucose level before surgery, but not by insulin treatment just before surgery. Both groups achieved a normal blood glucose level on day 3 and day 6 after surgery, and reduced the bacterial burden in the biceps femoris musle compared with that with no glycemic control.
Our study is the first study in which the effects of duration of preoperative insulin therapy on neutrophil functions were assessed before and after surgery. Our finding might be translate to human diabetic patients and generate the hypothesis that the duration of preoperative glycemic control would be influence to the postoperative outcomes in diabetic patients with chronic hyperglycemia. As there is still little information on this issue, our study would be relevant. Therefore, further discussion appears to be necessary.

Perioperative phagocytosis activity of neutrophils in diabetic mice and impact of preoperative insulin therapy
Chronic hyperglycemia is associated with dysfunction of neutrophil phagocytosis [7,8]. Surgical stress may induce further suppression of neutrophil phagocytosis activity [20][21][22]. In our study, chronic hyperglycemia lasted for about 6 weeks was associated with a 40% reduction in neutrophil phagocytosis activity. Surgical stress induced further 20-40% suppression of neutrophil phagocytosis activity in all four groups. In our model, preoperative insulin therapy for 5 days had restored the preoperative impairment of neutrophil phagocytosis activity, which was maintained after the operation. These results are in agreement with the results of the above-described previous studies [18,19]. However, short-term insulin therapy did not significantly improve neutrophil phagocytosis activity. There are several possible explanations for this finding.
First, inhibition of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway by chronic hyperglycemia has been reported to contribute to the suppression of neutrophil phagocytosis activity [23,24]. Such an impairment of the PI3K-Akt pathway is also associated with deterioration of insulin resistance, which may be restored by insulin therapy [24]. In the current study, ISF gradually improved and the improvement reached statistical significance after insulin therapy for 3 or more days. These results suggest that restoration of insulin sensitivity though various mechanisms including the PI3K-Akt pathway may require a certain duration of insulin therapy rather than single insulin administration. Since the PI3K-Akt pathway contribute to both insulin resistance and neutrophil phagocytosis, our diabetic model may require long-term insulin therapy for a significant improvement of neutrophil phagocytosis activity.
Second, in mouse bone marrow, promyelocytes grow into mature neutrophils during a period of 5 days. The mature neutrophils are pooled in bone marrow for 2 days and then released into blood.
Finally, neutrophils end up their life within 6 hours [25]. Since hyperglycemia would influence the glucose level in bone marrow, insulin therapy for 5 days may improve growth circumstance of mature neutrophils in bone marrow and then may contribute to the improvement of neutrophil phagocytosis.
Our results for the short-term group appear to conflict with the results of the study by Kroin et al. [19]. However, it should be noted that their short-term group received insulin treatment for 7 days after surgery and achieved a normal blood glucose after surgery. Then, they evaluated the bacterial burden at 7 days after the operation. Considering the fact, both the preoperative insulin therapy and postoperative insulin therapy may contribute to their results.
Perioperative ROS production activity of neutrophils in diabetic mice and impact of preoperative insulin therapy Hyperglycemia has been reported to induce activation of the protein kinase C pathway [24] and advanced glycation end products pathway [26], which may result in an increase in the production of ROS in neutrophils [27,28]. As was observed in db/db mice in Yano's study [18], our untreated diabetic group had significantly greater production of ROS than that in the non-diabetic group before surgery.
Although there was a trend for improvement in the production of ROS before surgery in the long-term group, short-term and long-term insulin treatment had no significant effect on perioperative neutrophilic ROS production in our study. The effect of insulin therapy on perioperative production of ROS may be influenced by various factors including the cause of diabetes [18]. Further examination is needed to clarify the relationship between duration of insulin therapy and changes in production of ROS by neutrophils.

Limitations
Several limitations of our study need to be considered. First, we did not conduct either blood glucose measurement or additional insulin therapy after surgery, which may have contributed to our results.
Second, we examined neutrophil functions only 24 hours after the operation. In future studies, the trend of postoperative neutrophil functions might be better to be examined. Finally, we did not assess the effects of hyperglycemia and insulin therapy on the intracellular signaling. The detailed signaling pathways describing the restoration of neutrophil phagocytosis with different durations of insulin therapy remain to be identified.

Conclusion
In our model in which chronic hyperglycemia was sustained for 8 weeks, preoperative and postoperative phagocytosis activities of neutrophils were restored by insulin therapy for 5 days before surgery but not by insulin therapy for 6 hours before surgery.    Doses of insulin administered each day. The dose of insulin administered on the first day in long-term group was not significantly different from that in the short-term group (p = 1.00).

Abbreviations
In the long-term group, the insulin dose required to control glucose level gradually decreased, and the insulin doses were significantly lower on the 3rd, 4th and 5th days of insulin therapy than on the first day (3rd, 4th, and 5th days vs. first day: p = 0.0001, < 0.0001, and < 0.0001, respectively).  Neutrophil phagocytosis rates before (A) and after (B) surgery in the four groups. (A) The phagocytosis rate in the short-term group was not significantly different from that in the untreated group (p = 0.87). The phagocytosis rate in the long-term group was significantly higher than that in the untreated group (p = 0.0008). The phagocytosis rate in the longterm group was comparable to that in the non-diabetic group (p = 0.63). (B) The phagocytosis rate in the short-term group was not significantly different from that in the untreated group (p = 0.41). The phagocytosis rate in the long-term group was significantly higher than that in the untreated group (p = 0.0005). The phagocytosis rate in the longterm group was comparable to that in the non-diabetic group (p = 0.14).

Figure 7
Reactive oxygen species (ROS) production before (A) and after (B) surgery in the four groups. (A) The median means fluorescent index (MFI) in the untreated group was not significantly different from that in the short-term group (p = 0.90) and that in the long-term group (p = 0.059). MFI in the non-diabetic group was significantly different from that in the untreated group (p = 0.0043). (B) The median MFI in the untreated group was not significantly different from that in the short-term group (p = 0.78) and that in the long-term group (p = 0.64). The median MFI in the non-diabetic group was not significantly different from that in the untreated group (p = 0.026).

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