Re: Lex Steele 4 Hours Long All Lexington All Anal
Loreta Mihor
Copyright: 2014 Starr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the National Institutes of Health grants (R01 AG039732). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Sepsis is a life-threatening clinical condition characterized by a profound systemic inflammatory response to infection. Progression to a more severe condition with shock, multiple organ failure, and death commonly occurs. Sepsis affects more than 700,000 people and claims at least 200,000 lives in the US annually [1]. This disease is particularly serious among the elderly population as both incidence and mortality drastically increase with advanced age [2].
Among various types of animal models for sepsis research, cecal ligation and puncture (CLP) is one of the most frequently used procedures to induce experimental sepsis in laboratory animals such as mice and rats [3], [4], [5]. Under the CLP model, polymicrobial peritonitis is induced in anesthetized animals by surgical ligation of the cecum followed by needle puncture to allow secretion of cecal contents into the abdominal cavity. The CLP model has been preferred by many investigators who study sepsis using laboratory animals because it is a relatively simple surgical procedure and closely mimics the clinical course of intra-abdominal sepsis [3]. The severity of CLP-induced sepsis is highly dependent on the degree of infection which can be influenced by volume, rapidity, and duration of cecal content released into the abdomen, and by the bacterial flora present in the cecum. Therefore, despite its popularity, CLP may not be preferable for certain experiments including those investigating animals with different cecum size, shape, or bacterial flora. This concern may apply for studies that compare severity of sepsis among animals with different body size (ex. neonatal mice or mutant dwarf mice), under different diet regimens (ex. liquid diet, high fat diet, or diet restriction), different gastrointestinal conditions (ex. neonatal or aged animals or animals with gastrointestinal pathology), or increased sensitivity to surgery (ex. aged animals or animals with deficient wound-healing capability).
In cases when CLP is not preferable, intraperitoneal injection of cecal slurry (CS) is an alternative method. In the CS model, contents from the cecum of sacrificed animal(s) are suspended in liquid form and injected into the abdominal cavity of other animals to induce polymicrobial sepsis [6], [7], [8], [9], [10]. The CS model of sepsis has been preferred by a limited number of investigators, particularly those who study sepsis in neonatal mice. However, one prominent disadvantage of the CS model is that the currently accepted protocol for CS preparation with 5% dextrose in water (D5W) does not allow for long-term storage, and thus the CS has to be freshly prepared each time an experiment is performed [11]. This can lead to significant variability from experiment to experiment. In the present study, to circumvent this problem, we developed a new CS preparation procedure that allows for the long-term storage of CS stocks without loss of bacterial viability. In addition, we validated survival rates of mice at different ages using stored CS prepared with this new protocol.
To induce chronic pancreatitis in mice, recurrent acute pancreatitis was induced in young mice (6-months old) by a procedure modified from our previous protocol for acute pancreatitis [12]. Each mouse received intraperitoneal (i.p.) injection with either physiological saline (control) or caerulein (American Peptide Company, Sunnyvale, CA) at a dose of 50 g/kg body weight, 6-times hourly, 3 days per week (Monday, Wednesday, and Friday) for 9 weeks. Body weight of each mouse was monitored daily. To induce diabetes, young (5-months old) fasted mice received streptozotocin (Sigma-Aldrich, St. Louis, MO) at a dose of 45 mg/kg body weight, i.p. once daily for 5 consecutive days. Control mice received no injection. Development of diabetes was confirmed by monitoring the non-fasted blood glucose level of each mouse by tail vein nick (ACCU-CHEK Nano blood glucose meter, Roche Diagnostics, Indianapolis, IN).
At sacrifice, ceca were dissected from mice using sterile instruments. Each cecum was placed in a plastic container, weighed, and photographed. The contents of each cecum were collected using sterile forceps and spatula, weighed (wet weight) and dried in an oven at 60C for 48 h (dry weight). A wet/dry ratio was calculated for the contents of each cecum.
Immediately after collection, cecal contents from young and aged mice were suspended in sterile water at a concentration of 5 mg/mL. The resulting suspension was mixed well and 50 L was spread onto multiple agar plates containing 3.7% w/v brain-heart infusion broth (211059, Becton, Dickinson and Company, Sparks, MD) and 0.15% w/v agar (214530, Becton, Dickinson and Company) with 8 mg/L aztreonam (Sigma-Aldrich) for Gram-positive bacteria selection [13], 105 units/L penicillin G (Sigma-Aldrich) for Gram-negative selection, or without antibiotics for total aerobic bacteria quantification.
Immediately after preparation of CS stock, an aliquot of CS was serially diluted with sterile saline and plated onto multiple agar plates containing 3.7% w/v brain-heart infusion broth and 0.15% w/v agar. CS aliquots stored at various temperatures for different periods of time were also diluted and plated in the same manner. The agar plates were incubated at 37C in ambient air for 24 hours before colonies were counted to determine colony formation unit (CFU).
To assess the extent to which CS injection induces endotoxemia, heat-inactivated CS was injected into the peritoneal cavity. A CS-containing vial was thawed and subsequently incubated at 72C for 10 minutes before administration to mice. A portion of the heat-treated CS was diluted and plated onto agar plates to confirm complete bacterial death.
To show that under some experimental conditions the CLP model of sepsis is not ideal, we compared cecum size, shape, and cecal content consistency in various animal models including diabetes, chronic pancreatitis, and aging. Type 1 diabetes was induced by multiple low dose streptozotocin (STZ) injection model (confirmed by hyperglycemia), and ceca were dissected from mice at sacrifice. Macroscopically, the ceca of diabetic and control mice looked similar in size and shape (data not shown). Cecal content wet weight were not different between the two groups (Fig. 2A), but cecal content wet/dry ratio (Fig. 2B) showed a significant difference indicating that fecal matter within the cecum of diabetic mice has a different consistency than normal mice. This could complicate CLP-induced sepsis because the watery cecal content of diabetic mice may leak into the abdomen from the puncture site much more rapidly than that of control mice.
Chronic pancreatitis was induced by repeated bouts of acute pancreatitis using caerulein injection and ceca were dissected from mice at sacrifice. Macroscopically, the ceca of mice with chronic pancreatitis appeared enlarged and fuller than that of control mice (Fig. 2D). Cecal content wet weight was significantly different (p
To test the efficacy of stored CS stocks for inducing sepsis, three different age groups of mice were intraperitoneally injected with standardized doses of CS and survival monitored for at least 10 days. The CFU of the injected CS was 4.7104/mL. As shown in Fig. 5A, 100 uL of CS was non-lethal to young and middle-aged mice but resulted in only 50% survival in aged mice. By increasing the dose of CS to 150 uL, survival in aged mice was reduced to 38%, survival in middle-aged mice was reduced to 67%, and young mice maintained 100% survival (Fig. 5B). A dose of 200 uL further reduced survival in aged and middle-aged mice to 0%, while young mice became susceptible with a 43% survival rate (Fig. 5C). Additional doses studied but not shown were 50 uL which resulted in a 63% survival rate in aged mice and 400 uL which killed all young mice.
(A) Blood was taken from the tail vein of young, middle-aged, and aged mice 12, 24, or 48 h after injection with 150 L CS and colony formation unit (CFU) assessed by spreading on agar plates. After fifteen days, data from mice which survived or died were separated into two groups and CFU compared. (B) Blood was taken from the tail vein of young mice 24 h after injection with 200 or 400 L CS and CFU assessed by spreading on agar plates. After ten days, data from mice which survived or died were separated into two groups and CFU compared. Each symbol represents data from a single animal.
To eliminate the possibility that death due to CS-induced sepsis occurs by endotoxic shock from bolus bacterial injection, an experiment was performed in which CS was heat-inactivated to kill bacteria and then injected to mice (Fig. 7). The same volume of vehicle (15% glycerol) and untreated CS were injected in parallel. Vehicle injection induced a very mild acute drop in body temperature of approximately 1.5C which returned to baseline within 12 h similar to our previous observation using saline as a vehicle [15]. Both heat-inactivated CS and untreated CS induced profound acute hypothermia with body temperature falling to 32C within 3 h after injection. Hypothermia was sustained at 32C in HI-CS injected mice for 24 h at which point the mice began to normalize and all survived, while the untreated CS-injected mice continued to exhibit profound and worsening hypothermia and eventually died. These results suggest that the CS model of sepsis induces non-lethal temporal endotoxemia, but that prolonged illness and death is due to infection.
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