18F-FDG-PET/CT in Initiation and Progression of Inflammation and Infection

Objective: Detection/localization of infection and inflammation is important for the initiation of correct treatment as well as its maintenance. Nuclear medicine imaging methods play an important role in determining infection and inflammation. 18F-2’-deoxy-2-fluoro-d-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) is highly sensitive in such cases when used with tomographic cross-sections. In this study, the development and progression of infection and inflammation were monitored on rats by using 18F-FDG via PET/CT. Methods: Sterile and infected abscesses were formed on rats using turpentine and S. aureus, respectively. For evaluation of the formation and progression of the abscess, 18F-FDG was injected into the rats and they were imaged by PET/CT at intervals of twenty-four hours for five days. Maximum standard uptake value (SUVmax) of 18F-FDG was calculated. Results: The highest activity involvement was seen on the first day of abscess formation. On the first day, SUVmax of the S. aureus abscess was 3.9±0.9 while in the sterile abscess SUVmax in the first day was 2.2±0.8. 18F-FDG uptake decreased day by day and it reached the background level on the fourth and fifth days. There were statistically significant differences between S. aureus and sterile abscess, and between sterile abscess and background activity in terms of SUVmax values during the first three days (p<0.05). On the fourth and fifth days, there was no statistically significant difference between S. aureus and sterile abscess, and between sterile abscess and background activity (p>0.05). Conclusion: The results demonstrated that the SUVmax value for 18F-FDG can be useful in the early differentiation of sterile and infected abscess. In addition, 18F-FDG-PET imaging has the advantage of local availability of equipment and labeled agents leading rapid diagnosis of differentiation of infection and inflammation.


Introduction
Detecting the presence and identifying the localization of infection and inflammation have primary importance for implementation of proper treatment and patient followup (1,2,3,4,5,6,7). Nuclear medicine offers powerful noninvasive imaging techniques for visualization of infection and inflammation-related disorders by imaging the whole body, thus enabling determination of both the localization and extent of inflammatory foci (8,9). Various methods have been developed that display different stages of the inflammatory response. Many radiopharmaceuticals have been evaluated extensively in both preclinical and clinical studies as potential diagnostic agents to identify the sites of infection (10,11,12,13). Although there are several imaging agents, only a few of them are being used in routine clinical practice. There is a definite role of 18 F-2'-deoxy-2-fluorod-glucose ( 18 F-FDG) in assessing disease extent, disease activity in patients with infection and inflammation, and evaluation of response to treatment (6,14). The high tissue radioactivity after administration of 18 F-FDG corresponds to increased glucose uptake and consumption through the hexose monophosphate shunt, which is the main source of energy for chemotaxis and phagocytosis (7,15). 18 F-FDG, an analog of glucose, is taken up by living cells via cell membrane glucose transporters and subsequently it is phosphorylated with hexokinase inside most cells. Activation of phagocytes, also known as respiratory burst activation, lead to increased 18 F-FDG uptake (10). In sterile inflammation, administered 18 F-FDG is mainly taken up by neutrophils and macrophages (6,15). A high degree of 18 F-FDG uptake is detected in neutrophils during the acute phase of inflammation, while macrophages and polymorphonuclear leukocytes uptake 18 F-FDG during the chronic phase (1,6,11,16). 18 F-FDG is phagocytized by macrophages and phagocytic cells via d-glucose transporter. Through glycolysis, 18 F-FDG is phosphorylated by hexokinase resulting in 18 F-FDG-6 phosphate. Positron emission tomography (PET) imaging can be used alone or in conjunction with computed tomography (CT) in diagnosing and management of therapy planning in a variety of disorders (9). The use of PET/CT represents the new generation in diagnostic modality. PET imaging detects an increase in metabolic activity while CT provides anatomic correlation (15,16,17,18). Inflammatory cells have an increased positive expression of glucose transporters and growth factors, which affect the affinity of these transporters for deoxyglucose. Due to structural analogy, 2-deoxyglucose 18 F-FDG is uptaken at the site of infection at a high level depending on the rate of glycolysis. 18 F-FDG is carried into the cells by the glucose transporters. In case of infection and inflammation, leukocyte activation occurs and glucose is used in the activation as an energy source. Glucose transporter receptors are stimulated by uptake of glucose and its analogues (17,19). The aim of this study is to evaluate the development of infection and inflammation, as well as to monitor sterile and infected abscesses in rats by using 18 F-FDG-PET/CT.

Materials and Methods
All animals were treated in accordance with the protocols approved by the Animal Care and Use Committee of the University. The designed study was conducted at the animal care facility of the Faculty of Medicine, Dokuz Eylül University. In this study, sterile abscess was induced by using turpentine and infected abscess was induced by using Staphylococcus aureus ATCC 25923 strain on rats. Three groups of rats were used for imaging as sterile, infected, and control groups. Another group of rats were used to remove the abscess tissue to compare number of living organism with standard uptake value (SUV). They were male White Wistar Rats, clinically healthy animals of 150-220 gr body weight.

Bacterial Strain and Rat Model for Abscess Formation
S. aureus strain (ATCC 25923) was grown in 5-10% sheep blood agar (Salubris, USA) after incubating overnight at 37°C. One loop of the S. aureus colonies was suspended in Mueller Hinton broth (mhb) containing tube to obtain l0 7 colony-forming units (CFU)/mL. The animals were anesthetized by intraperitoneal administration of xylazine, 5 mg/kg and ketamin, 35 mg/kg. For infected abscess formation on rats (n=14), S. aureus 0.5 mL 10 7 CFU/mL was inoculated in the right arm of the rats subcutaneously. For sterile abscess formation on rats (n=7) 0.2-0.4 mL turpentine (Sigma-Aldrich) was injected into the right arm of the rats subcutaneously. In the control group (n=6), 0.5 mL 0.9% NaCl was injected into the right arm of the rats subcutaneously. Following each imaging, an abscess of a rat was removed in the another S. aureus group and living bacterial organisms were counted in the excised tissue.

Positron Emission Tomography/Computed Tomography Imaging of Rats
A preliminary study was performed to optimize imaging time depending on abscess formation. First day image was acquired 24 hours after inoculation of S. aureus and turpentine. 18 F-FDG (37 MBq) was injected intravenously via the tail vein. Prior to 18 F-FDG injection, rats fasted for 4 hours and were well hydrated. Imaging was performed using PET/CT (PHILIPS Gemini TF), beginning one hour after injection of 18 F-FDG on the first day for five days with an interval of twenty-four hours. 18 F-FDG rat imaging was done with two-minute bed positions. On the first day, imaging was performed at the first and second hours after injection of 18 F-FDG to obtain optimum imaging time. Non-diagnostic CT images were obtained (90 kVp and 30 mAs, with a thickness slice of 2 mm, the rotation time was 0.5 sec, 39 mm/sec bed speed, 512x512 matrix).

Image Analysis
PET/CT images were visually and semi-quantitatively assessed. For semi-quantitative analysis of the PET images, a region of interest (ROI) was drawn around the abscess area on the right arm. In the control group, a ROI was drawn around a similar area as the background on the right arm. SUV max was obtained from the images for evaluation of glucose metabolism of infection and inflammation detected by 18 F-FDG-PET/CT. The SUV max of 18 F-FDG uptake were calculated on abscess sites by using the formula: SUV=Tissue concentration (Bq/g)/[injected dose (Bq)/body weight (g)]

Results
Twenty-four hours after inoculation of S. aureus or turpentine, swelling was apparent in the abscess site. A soft tissue infection developed on the right arm within twentyfour hours after bacterial inoculation. Swelling and redness of the abscess area were apparent in all rats. Abscess sites were visualized by 18 F-FDG-PET/CT. A higher abscess/ background ratio was detected at the first hour compared to the second hour after injection of 18 F-FDG. Imaging time was chosen as the first post injection hour for the following days. The initial SUV max for S. aureus was 3.9±0.9 on the first day, while it was 2.2±0.8 for sterile abscess and 1.2±0.5 for control group rats. The first day SUV max on the second hour following 18 F-FDG injection was 2.8±0.6 for infected abscess, 1.9±0.9 for sterile abscess, and 1.2±0.09 for control group. During the following two days, although activity involvement decreased at S. aureus it was still higher than involvement in turpentine abscess. There were statistically significant differences between S. aureus and sterile abscess, and between sterile abscess and control group as SUV max for the first three days (p<0.05). On the fourth and fifth days, there was no statistically significant difference between S. aureus and sterile abscess, and between sterile abscess and control group (p>0.05). It was observed that SUV max of infected abscess was higher than that of sterile abscess in all images during the first three days. CFUs per milliliters (CFU/mL) in excised abscess tissue was 10 4 CFU/mL, 10 3 CFU/mL, 10 2 CFU/mL in the first, second and third days, respectively. There was no living organism at the fourth and fifth days. A correlation was detected between 18 F-FDG activity and number of living microorganisms in excised abscess tissue. Figures 1a, 1b, 2a, 2b display 18 F-FDG images of sterile and infected abscess in rats. Table 1 presents SUV max for sterile abscess, infected abscess, and control rats as well as the quantity of living organisms.

Discussion
It is clinically important to distinguish infection from inflammation (6,14). Abscess is a life threatening and important complication of inflammation or major surgery (19,17). Most infectious and inflammatory foci can be visualized accurately with radiolabeled autologous leukocytes. In vitro labeled leukocyte imaging is the gold standard for imaging most infection. However, preparation of this radiopharmaceutical is laborious, time consuming and requires handling of potentially contaminated blood (4). New agents are being developed that could potentially differentiate between infection and non-microbial inflammation. In addition to these, it is suggested that 18 F-FDG-PET imaging can be used to visualize inflammatory foci when a high spatial resolution is required (1). Kumar 18 F-FDG uptake and its distribution in turpentine induced inflammatory tissue on male Donryu rats. They showed that the uptake in inflammatory tissue increased gradually upto 60 minutes and then decreased. Our study also showed an increased activity within 60 minutes followed by a decreased activity 120 minutes after injection of 18 F-FDG. They reported an increasing activity which peaked on the 4 day after inoculation followed by a slow down. Our study detected the highest 18 F-FDG activity a day after turpentine inoculation. In our study, there was also a correlation between 18 F-FDG activity and quantity of living microorganisms in excised abscess tissue. These differences may be due to the rat's immune tolerance. Kaim et al. (22) studied 18 F-FDG and 18 F-FET in an acute phase abscess model. Their histological study showed increased 18 F-FDG uptake that corresponded to cellular inflammatory infiltrates, mainly consisting of granulocytes. The necrotic abscess center and the second necrotic tissue layer were characterized by decreased 18 F-FDG uptake. They documented a marked increase in 18 F-FDG uptake at the site of infection, which could be attributed to activated granulocytes and macrophages. In their study, 18 F-FET uptake was low in inflammatory infiltrates consisting of neutrophil, granulocytes and macrophages. They interpreted this finding as either low uptake in the bacterial area may be lacking bacterial uptake by 18 F-FET or small number of living bacteria in the infection area. On the other hand, our study demonstrated that there was a correlation between bacterial load and 18 F-FDG uptake rate. A greater bacterial load in the excised abscess yielded a higher SUV. Sugawara et al. (23) compared 18 F-FDG, thymidine, L-methionine, 67 Ga Citrate and 125 I-HSA in sites of bacterial infection in rats infected with E. coli. Their auto-radiographic study detected the highest 18 F-FDG uptake in the inflammatory area of cell infiltration surrounding the necrotic region. In their study, 18 F-FDG showed much higher uptake values than 67 Ga Citrate or 125 I-HSA. They reported lower methionine and thymidine accumulation in the infectious foci than 18 F-FDG. In their bacterial model, an abscess was formed and the necrotic area showed slightly higher 18 F-FDG uptake than the surrounding edematous muscle, while the center of the abscess in turpentine model showed very low 18 F-FDG uptake. In our study, 18 F-FDG uptake in the bacterial site was higher than the inflammation site. It has been reported that abscess-forming bacteria utilize glucose as an energy source using various pathways. The lack of any 18 F-FDG uptake could be attributed to a low number of inflammatory cells, lack of granulation tissue, or absence of microorganisms.  In our study, the highest 18 F-FDG activity was observed on the first day following inoculation in the infection and the inflammation sites, and then the activity decreased day by day. This could be related to the rapid metabolic activity in fast recovering rats. On the fourth and fifth days, 18 F-FDG activity was equal to that of the controls. We compared activity rates according to bacterial load by excising tissue. It was seen that bacterial load and 18 F-FDG uptake values correlated with infection severity. In this study, the imaging was performed by PET/CT that is designed for human body imaging, if imaging studies had been obtained with small animal imaging device (microPET/CT) resolution of the images would have been higher than the presented images.

Conclusion
Inflammatory and infected tissues utilize glucose as energy source for chemotaxis and phagocytosis. Therefore, the accumulation of 18 F-FDG as a glucose analog can be used for diagnosis of infection and inflammation. Sterile and infected abscess differentiation can be evaluated by imaging with 18 F-FDG-PET at early phase. The value of SUV max explores correlation between sterile abscess and infected abscess. 18 F-FDG-PET is also a useful technique to understand the extent of the infection and inflammation process. In addition to PET imaging method with 18 F-FDG has the possibility of rapid diagnosis and easier with the advantage of local availability. Ethics

Ethics Committee Approval and Informed Consent:
All animals were treated in accordance with the protocols approved by the Animal Care and Use Committee of the University. The designed study was conducted at the animal care facility of the Faculty of Medicine, Dokuz Eylül University. Peer-review: Externally peer-reviewed.

Authorship Contributions
Surgical The authors declared that this study received no financial support.