Abstract
Intravital two-photon microscopy (2PM) is an advanced fluorescence based imaging technique that allows for a cinematic study of physiological events occurring within tissues of the live animal. Based on this real-time imaging platform, the pathophysiology of bacterial infections can be studied in the most relevant of model systems—the live host. Whereas traditional animal models of host–pathogen interaction studies rely on end stage analysis of dissected tissues, noninvasive intravital imaging allows for real-time monitoring of infection during shorter or extended time frames. Here we describe the use of advanced surgical techniques for initiation of spatially and temporally well-controlled kidney infections in rats, and how the bacterial whereabouts can be studied while at the same time monitoring the host’s altered tissue homeostasis based on real-time deep tissue imaging on the 2PM platform. Whereas this chapter focuses on pyelonephritis induced by uropathogenic Escherichia coli (UPEC) in rats, the major concepts can easily be translated to numerous infections in a variety of organs.
Key words
1 Introduction
The establishment of the field “cellular microbiology” during the 1990s has been pivotal in our current understanding of how pathogens interact with their hosts during infection. The generated wealth of knowledge, to a large part based on molecular in vitro studies, has essentially molded our understanding of infection biology and guided the development of antimicrobial therapies. However, the true state of an infection within the living host is fundamentally complex and changes over time as infection progresses, sometimes reaching deeper tissues or disseminating into the circulation of the animal, causing sepsis. To fully understand the infection process thus calls for non-reductionistic approaches applicable to the live animal.
The organs are highly complex, comprising a number of different tissues acting in concert to achieve the physiological function of the organ. The kidney is represented by a heterogeneous organization of nephrons, blood vessels, and connective tissues, comprising various cell types of epithelial, endothelial, and immune cell origin. This translates to a range of unique microenvironments in close proximity to each other, where infecting bacteria may reside during the different phases of infection. Depending on the tissue’s histology, anatomy, and physiology, the infected organ may communicate with proximal and/or distal organs when mounting a proper response to a local infection. Intravital imaging has revealed the nature of several physiological events that are closely associated with UPEC infection of the proximal tubules in the renal cortex, as exemplified by gradual cessation of renal filtration leading to nephron obstruction and glomerular shutdown [1]. Localized ischemia ensues with tissue oxygen tension dropping rapidly, thus establishing an anaerobic bacterial microenvironment already a few hours into the infection. The ischemic response is in part dependent on activation of the clotting cascade in peri-tubular capillaries, and this was shown to serve as a protective innate mechanism. By containing bacteria at the site of infection, spread to deeper tissues and eventually the systemic circulation is avoided during the neutrophil recruitment phase. To maintain their foot hold inside the tissue in spite of the dynamic alterations in tissue histology and homeostasis, it is very likely that bacteria adapt their metabolomic and proteomic profiles throughout the infection process.
The complexity of infection-associated tissue alterations thus drives the need for novel model systems, which takes the relevant host and microbe complexities into account. As studies based on intravital imaging of infection within organs of live animals forms the basis of the emerging area “tissue microbiology,” this field has the potential to deepen our understanding of the integrated pathophysiology of infections [2–4].
Fluorescence-based microscopy of host–pathogen interactions in vivo was for long hampered by several factors, a major being the limited depth penetration of light as it is absorbed and/or diffracted by tissues. Moreover, some of the most commonly used routes of infecting an animal (intravenous, intraperitoneal, intragastric, bladder catheterization) results in poor spatial and temporal predictability of the progression of infections. This is exemplified by the ascending model of pyelonephritis: following infusion of bacteria into the urinary bladder, it is impossible to foresee which population of the millions of nephrons bacteria will colonize at any point in time. The creative use of advanced surgical techniques for time-controlled bacterial delivery has thus been critical in allowing for high-quality visualization of infection processes within precise locations in the organ of a host animal [5–8].
2PM was originally developed by biophysicists [9] and later adapted to the live studies of dynamic processes in organs by biologists [10]. In essence, reduced phototoxicity and deeper tissue penetration of the excitation light is achieved by the use of pulsed beams of photons possessing half the energy required to excite a fluorophore. A detailed review of the technical aspects of 2PM can be found in ref. 11. 2PM has been applied to multiple medical fields to study processes in a wide array of intact tissues. Non-exclusively, these include the study of calcium fluctuations in individual synapses [12, 13], the role of astrocytes in the brain [13], tumor vascularization [14], embryonic development [15], kidney physiology [16], immune cell homing in inflammation [17, 18], and bacterial infections of the kidney [8, 11, 19, 20].
The applicability of 2PM in studies of bacterial infections is in this chapter exemplified by detailing the procedure for real-time intravital imaging of uropathogenic Escherichia coli (UPEC) infection of initially one nephron as a model for pyelonephritis. Many considerations of the procedures are highly generic, and our aim is that this chapter may serve as a guideline for details to be considered by scientists planning to initiate an intravital imaging model for a bacterial infection. The following sections cover detailed descriptions of the setup of intravital 2PM imaging of the kidney, surgical procedures for initiation of infection, as well as generation of the fluorescent probes used to visualize important components at the site of infection.
To visualize bacteria by 2PM imaging, bacteria should express a fluorescent protein, such as GFP+. Preferentially, the protein should be encoded from a stable, single insertion of the gene segment on the chromosome rather than a plasmid to avoid the need of antibiotics in the animal during the time-course of infection [8]. The choice of promoter is also relevant. To circumvent altering levels of bacterial fluorescence due to changes in the local tissue environment, a constitutively active promoter can be used, such as the tetracycline promoter PLtetO-1 [8].
To obtain the spatial and temporal precision required for the dynamic 2PM imaging, surgical procedures can be applied when initiating infection. In the pyelonephritis model, access to the left kidney of a Sprague Dawley rat was achieved by gentle, surgical exposure. Using a fine glass capillary needle, bacteria can be slowly infused into the tissue, with care taken to avoid introducing unnecessary damage to the tissue. In the pyelonephritis model, 105 CFU of the UPEC strain CFT073 is slowly infused into the lumen of a single nephron with an infusion rate and pressure corresponding to the native rate of glomerular filtration. A distant site injected with phosphate buffered saline according to the same principle acted as a sham-operated control site within the same kidney of the anesthesized animal.
Within a live host, the local infection may traverse into or induce responses in several tissue compartments. Tissue autofluorescence originating from a specific subset of cells, e.g., proximal tubular cells, can be used as an advantage to help in the tissue orientation. To highlight the vasculature, fluorophore-conjugated dextrans can be systemically applied [6, 7, 16]. Dextrans above the size-exclusion limit (i.e., 50 kDa dextran) cannot be filtered by the glomerulus and thus remain in circulation. In contrast, small molecular weight dextran (10 kDa) injected into the systemic circulation is rapidly filtered, and can accordingly be used to visualize glomerular filtration and determine filtrate flow rate in the tubular segments of the nephron. If bacteria are introduced into compartments exposed to significant shear stress, such as the renal filtrate in the tubules of the nephron, the bulk of infused bacteria will be flushed away, whereas only few remain in the tissue from where the infection is initiated. To aid in identification of the infected nephron, a fluorophore-conjugated small molecular weight dextran (10 kDa) can be co-infused with the bacterial inoculum. Due to endocytic activities of proximal tubule cells, the fluorophore-conjugated dextran will serve to provide a distinct outline of the apical side of the injected epithelium [8].
2 Materials
2.1 Bacterial and Culture Requirements
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1.
UPEC strain LT004 (cobS:: ϕ(PtetO-1) gfp + Cmr) [8] (see Note 1 ).
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2.
Luria–Bertani (LB) agar and LB broth.
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3.
37 °C shaking incubator for liquid culture growth.
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4.
37 °C incubator for agar plates.
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5.
Inoculating loops.
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6.
100-mL conical flask or 15-mL Falcon tube.
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7.
Centrifuge.
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8.
1.5-mL Eppendorf tubes.
2.2 Fluorophore Conjugated Dextrans
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Sterile 0.9 % saline solution.
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2.
10,000 MWCO membrane.
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3.
Surgical syringe.
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4.
10 kDa fluorophore-conjugated dextran in 0.9 % sterile saline (20 mg/ml), store wrapped in foil ≤ 1 month at 4 °C.
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5.
500 kDa rhodamine-conjugated dextran in 0.9 % sterile saline (8 mg/ml), store wrapped in foil ≤ 1 month at 4 °C. Dialyze probe solution (5–10 ml) before use against 0.9 % (w/v) sterile saline (5 L) overnight at room temperature using a 10,000 MWCO membrane (see Note 2 ).
2.3 Intratubular Microinjection Induced Infection
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1.
Standard Sprague–Dawley (264 ± 16 g) and Munich-Wistar (255 ± 22 g) male rats. Animal experimentation should follow the local ethical and legal national regulations and be performed by trained individuals accredited by the relevant regulatory bodies.
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Anesthesia induction chamber.
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3.
Isoflurane–oxygen mixtures (5 % (v/v) and 2 % (v/v)).
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Halothane–oxygen anesthesia.
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Pentobarbital (optional).
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Buprenorphine.
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7.
50 mm dish and 40 mm coverslip.
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8.
Autoclave tape.
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9.
Appropriate animal temperature control devices (e.g., circulating water blanket attached to a temperature-controlled circulating water bath).
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10.
ReptiTherm pads.
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11.
Homeothermic table.
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12.
Rectal probe for temperature recording.
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13.
Vascular catheters (PE-60 tubing for rats and PE-50 tubing for mice).
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14.
Electric clippers.
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Germicidal soap.
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Surgical scissors.
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A pair of tooth forceps.
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18.
A pair of hemostats.
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19.
Stereoscopic microscope.
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20.
Sharpened micropipettes (5–10 μm inner diameter). Pulled using a micropipette puller and sharpened on a wet spinning grindstone at a 20o angle.
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21.
Leitz micromanipulator.
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22.
Micropump.
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23.
Kidney cup.
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Sterile 0.01 M phosphate buffered saline prewarmed to 37 °C.
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25.
Heavy mineral oil.
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Sudan Black-stained castor oil.
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27.
WillCo-dish coverslip bottom dishes; 50 mm/40 mm coverslip (Electron microscopy sciences).
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28.
Appropriate fluorescent probes.
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29.
Imaging Platform.
3 Methods
3.1 Presurgery Preparation and Maintenance of Bacterial Cultures
Prior to the experiment, bacterial cultures are maintained on LB agar plates (see Note 3 ).
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A day prior to the experiment, prepare a fresh overnight culture of bacteria by inoculating one defined colony into 4 ml of LB broth in a 15 ml Falcon tube, incubate overnight shaken at 230 rpm at 37 °C.
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2.
On the day of the experiment, prepare a fresh culture by pipetting 40 μl of the overnight culture into 4 ml of fresh LB broth in a 15 ml Falcon tube.
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3.
Cultivate this culture at 230 rpm, 37 °C until the culture density reaches OD600 = 0.6.
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4.
Harvest the bacteria by centrifugation at 5,000 g for 5 min.
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5.
Discard the supernatant and wash the pellet twice with 0.9 % sterile normal saline. Concentrate the culture to an approximate density of 109 CFU/ml (see Note 4 ).
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6.
Store this suspension on ice for use within 2 h.
3.2 Surgical Preparation of Animals
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Apply anesthesia to the animal by placing it in an anesthesia induction chamber infused with 5 % isoflurane–oxygen.
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2.
After sufficient effect, transfer the animal to a clean heated surgical area (e.g., a homeothermic table).
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3.
Supply a 2 % isoflurane–oxygen mixture to maintain anesthesia. Titrate when necessary for effect.
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4.
Perform a subcutaneous injection of 0.05 g/kg buprenorphine.
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5.
Prepare the areas for incision by shaving the fur using a pair of electric clippers. Areas include left flank area (kidney exposure), neck (jugular vein and artery access), and inner thigh (femoral vein).
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6.
Disinfect the respective areas with germicidal soap and water.
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7.
Dry the areas with a paper towel and thoroughly clear away any remaining cut hairs (see Note 5 ).
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8.
Constantly monitor the temperature of the animal by inserting a rectal probe while awaiting the next step of the experiment (see Note 6 ).
3.3 Rat Surgery and Bacterial Infusion
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1.
Prepare the inoculum for microinjection. At least two micropipettes need to be prepared, the first is loaded with the bacteria culture, and a second filled with 0.9 % NaCl which functions as the sham (see Notes 7 – 9 ).
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2.
Prepare an optional third micropipette by aspirating Sudan-Black castor oil. Castor oil remains in the nephron when injected. When introduced to nephrons neighboring the infection site, this becomes a visual marking for the easy location of the infection site (see Note 10 ).
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3.
Begin preparing the animal for infection by first inserting a venous access line by making a small incision above the femoral vein.
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4.
Make a small cut in the vessel, insert a PE-60 tube and secure with sutures.
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5.
Once all preparative steps have been completed, locate the kidney and estimate its size by palpating the left flank of the animal.
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6.
Grasp the skin of the animal with a pair of tooth forceps.
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7.
On the intended path of incision, pinch the skin to crush the tissue with a pair of hemostats to prevent bleeding.
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8.
With a pair of surgical scissors, perform 0.5–1 cm incisions through the tissue. Repeat steps 6–8 for the outer muscle layer to expose the inner muscle layer.
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9.
At this point, re-palpate the tissue to locate the kidney.
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10.
Repeat the steps 6–8 again to gain access to the peritoneal cavity. Initial incisions should be shorter than the length of the kidney. The incision can be increased subsequently if needed (see Note 11 ).
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11.
Remove the adipose tissues encapsulating the kidney by careful manual searing with a pair of forceps (see Note 12 ).
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12.
Transfer the animal to a clean heated surgical area on the stereoscopic microscope stage.
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13.
Gently raise the kidney out of the peritoneal cavity by grasping the hilar fat pad with forceps.
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14.
Place the kidney in a kidney cup and stabilize the setup.
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15.
Supply normal saline through the femoral access, and add drops over the exposed kidney to maintain hydration. This should be performed throughout all subsequent steps.
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16.
Switch the anesthesia supplied to the animal from the original isoflurane–oxygen mixture to halothane–oxygen anesthesia. This allows for fine adjustments of anesthesia depth to be made, as well as recovery.
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17.
Shift the animal onto the stereomicroscope stage.
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18.
Illuminate the intended site of infection with a mercury levelling bulb. Focus at this site and increase the magnification to 96× (Fig. 1 ). Adjust the lighting where necessary (see Note 13 ).
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19.
Focus the field of view slightly about the kidney surface such that the kidney is now blurred.
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20.
Mount the bacterial suspension-filled micropipette onto the Leitz micromanipulator.
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21.
Bring the tip of the micropipette into the field of view just above the kidney (see Note 14 ).
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22.
Refocus onto the kidney surface.
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23.
Position the needle tip above a target nephron. The orientation of the needle should be aligned with the tubular walls. Approximately 90 % of surface-localized tubules are proximal convoluted tubules.
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24.
Advance the needle slowly into the proximal convoluted tubule until the needle tip breaks the tissue barrier and enters the luminal space. Upon contact with the tissue, the kidney capsule will offer a sizable degree of resistance (see Notes 15 and 16 ).
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25.
Infuse the bacterial suspension at a rate of 50 nl/min for 10 min, giving a total injected amount of approximately 5 × 105 CFU of bacteria.
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26.
Withdraw the needle from the tissue (See Note 17 ).
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27.
Replace the current micropipette with the Sudan-Black stained Castor oil filled micropipette (see Note 18 ).
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28.
Repeat steps 16–20 to make two marks on either side of the injection site for subsequent orientation on the 2P microscope stage.
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29.
When the microinjection has been completed, gently place the kidney back into the peritoneal cavity.
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30.
At this point, the animal may be used for short (below 8 h) and long term (above 8 h) imaging and/or non-imaging analyses (see Note 19 ).
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31.
For immediate imaging, transfer the animal to the 2PM stage (see Subheading 3.4).
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32.
Alternatively, for imaging at later time points or non-imaging analyses, close up the animal by suturing the retroperitoneum.
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33.
Single house the animal in the home cage and allow for recovery with sufficient food and water.
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34.
The kidney can be re-exposed for imaging at a later time.
3.4 Imaging Procedures
Numerous variants of 2PM imaging systems are available, and as each system has its own settings and nuances, the settings on the optics will not be discussed in detail here. Rather the reader is referred to [5, 6] (see Note 20 ). The system described in the methods below utilizes an inverted imaging system.
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1.
Prepare a raised scaffold inside a dish (50 mm dish with a 40 mm coverslip in the bottom) to stabilize the kidney on account for its curved surface. This can be achieved by stacking 4–7 pieces of 2 cm long strips of autoclave tape at the edge of the coverslip. Take care not to block the objective’s light path.
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2.
To maintain the temperature of the animal, place 2 ReptiTherm pads on each side of the dish and a warming jacket blanket over the stage.
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3.
Position the rat such that the ventral side of the kidney contacts the base of the dish. The curvature of the kidney should be stabilized by the scaffold made in Subheading 3.4, step 1. Flood the dish with 0.9 % saline to maintain hydration of the exposed organ.
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4.
Check for motion in the field of view under the 10× or 20× objective. If significant motion is observed, adjust the rat to further stabilize the kidney (see Note 21 ), e.g., positioning thorax of the rat away from the coverslip bottom with the kidney close to the edge. Take notice to avoid hyperextending the vessels. An example of an infected nephron is shown in Fig. 2 .
3.5 Post-imaging Procedures
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1.
Upon completion of imaging and/or of experimentation: sacrifice the animal according to relevant local animal handling directives.
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2.
Streak 100 μl of blood, collected for example by the heart puncture method, on an LB agar plate and incubate at 37 °C overnight to analyze whether infection remained local or if systemic spread occurred.
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3.
Further analysis of bacterial dissemination can be performed by plating homogenates from biopsies or from organs, such as the liver and spleen (See Note 22 and 23 ).
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4.
The bacterial population present at the renal infection site can be estimated after isolating the tissue with a 5 mm biopsy punch, and subsequently performing colony counts on the homogenized tissue.
4 Notes
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1.
The use of a chromosomally located, constitutive active promoter that regulates the reporter gene expression is essential to (1) avoid use of antibiotics in the animal to maintain the plasmid, and (2) ensure constant reporter (GFP+) expression in the microenvironment [20].
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2.
Most dextran preparations are polydisperse, containing a range of dextran sizes. Dialyzation is essential to ensure that no small molecular weight molecules are present which become filtered into the nephrons.
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3.
Maintenance on agar plates also allows for easy identification of contamination of the culture.
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4.
Repeated washing steps are recommended to remove immunogenic culture debris such as lysed bacterial components and LPS.
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5.
The presence of hairs severely reduces the image quality during the subsequent 2PM imaging. Hairs present as intense cylindrical shadows.
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6.
Constant monitoring of body temperature is essential as the temperature of the animal can drop drastically during surgical preparation of the animal as well as during imaging.
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7.
Extreme care has to be taken with the micropipettes. The sharpened tips are highly brittle and break easily. If chipped or broken, they are more likely to cause tissue damage rather than a clean infection.
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8.
The inoculum may be introduced into the micropipette by either drawing the respective solution from the tip, or via a PE-60 tubing and syringe from the back end of the pipette.
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9.
Do not allow the presence of air bubbles within the inoculum segment of the micropipette.
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10.
Crystallized forms of Sudan Black require a day or two to completely dissolve in castor oil. In addition, the solution needs to be filtered before use (Also see Notes 8 and 9 ).
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11.
To avoid motion of the kidney during the 2PM imaging procedure, incisions should be made as small as possible, but can be enlarged if required.
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12.
Adipose tissue is highly autofluorescent and must accordingly be removed at positions, which would affect imaging. However, it is important to leave a few patches as sites to safely adjust the kidney with forceps.
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13.
Take caution not to overexpose the kidney to light; lamps may emit heat which can desiccate and damage the tissue.
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14.
The infection site must be carefully chosen to ensure optimal imaging. Ensure that the site contacts the glass surface of the dish just above the microscope objective in the inverted microscope setup. The kidneys, as most organs, have a good degree of curvature, which will impede the imaging process if infection site is not carefully chosen.
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15.
Upon encountering resistance from the kidney capsule, advance the needle in short intermittent pauses. The breaking of the capsular layer will be sudden to which the needle may puncture through both walls of the tubule.
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16.
Hydration of the kidney should be liberal (see Subheading 3.3, step 15). If dehydrated, the kidney capsule becomes plastic-like and no longer offers a firm resistance to the micropipette tip. Instead, application of the micropipette will result in deep depression of the kidney without penetration of the capsule. In this event, when the capsule is finally penetrated, the micropipette will stab far deeper beyond the intended surface located tubules.
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17.
If microinjection has punctured or was performed mistakenly into a blood vessel, the withdrawal of the needle will be accompanied by the release of blood on to the kidney surface. Perforation of the blood vessel in this step results in a non-local infection, which contradicts the aim of this model.
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18.
If more than one set of micromanipulators and micropumps is available, infusion of bacterial inoculum and Sudan-Black castor oil may be performed simultaneously.
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19.
The measure of tissue oxygen tension is an example of non-imaging analysis which we have performed. The procedure involves first the setting up of a tail cuff or direct arterial line with a pressure transducer to monitor blood pressure throughout the procedure. This is followed by performing a two point calibration of the Clark type electrodes with either Na2S2O5 saturated H2O or in air at 37 °C. After the infections with UPEC and sham have been initiated, insert one microelectrode into each tubular lumen while working under the stereoscopic microscope. Readings can then be collected for the desired duration, to which the data should be presented showing the comparison of the PO2 at both infected and sham sites along with the blood pressure within the time frame of the experiment.
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20.
We would instead like to draw attention to the design of the imaging system, more specifically whether the optic system is upright or inverted. This is critical as the animal preparation differs greatly between the two systems. In the upright system, the exposed kidney is placed in a kidney cup held above the exposed peritoneal cavity. To accommodate the associated fixtures, the incisions are typically larger. Whereas unwanted tissue motion is minimized in the upright system, the tendency for mortality is increased. In the inverted system, which is described below, the rat is positioned on its left side, with the kidney dipped into a saline filled petri dish placed over the objective. The kidney will be inherently stabilized since the weight of the animal is positioned over the organ (Fig. 1). Unwanted movements are thus kept to a minimum.
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21.
Motion is highly detrimental to live imaging. However, there is a limit to the extent of tissue-stabilizing surgical procedures that can be performed without resulting in mortality. One should also keep in mind that a basal level of motion is unavoidable. This includes motion due to breathing as well as pulsations from the heart and systemic circulation. If problem with motion, one may use 2PM systems with high image capture rates, or apply post imaging software that compensates for image drift. Our system is custom designed with a capture rate of one frame per second for a 512 by 512 pixel image and one frame per 2 ms for a line scan.
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22.
A key feature of the intravital model described here is the high degree of spatial and temporal precision. We have found this to promote an exceptional reproducibility between experiments in the live setting. Knowing the exact position of the infection site allows for analyses by other techniques to supplement the imaging data (see Note 1 ). Alternatively, the dynamic real-time monitoring of the infection site can be combined with endpoint studies since tissue biopsies containing the foci of infection can be accurately obtained. The biopsy can be used for other immunohistochemical analyses, or for transcriptomics analyses (see Note 23 ). The precise dissection of the infection site minimizes any dilution from uninfected tissue, thus allowing for total RNA extraction and transcriptomic analysis. Collectively, the multiple data sets that can be obtained and combined with real-time 2PM imaging aid in creating the full picture of the integrated pathophysiology of infection.
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23.
Accurate isolation of the infected tissue is highly beneficial for RNA extraction and microarray studies. The spatial control of our model allows for the amount of uninfected tissue in the biopsy to be minimized, which greatly increases the possibility of capturing infection-specific molecular details in the transcriptomics assays. The temporal control of our model enables harvesting of biopsies at well-defined time points, resulting in a precise description of the host response during infection [21].
Abbreviations
- 2PM:
-
Two-photon microscopy
- UPEC:
-
Uropathogenic Escherichia coli
- GFP:
-
Green Fluorescent Protein
- CFU:
-
Colony Forming Unit
- PO2 :
-
Tissue oxygen tension
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Acknowledgement
The research relevant to this chapter was supported by the Swedish Research Council and the Swedish Medical Nanoscience Center.
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Choong, F.X., Richter-Dahlfors, A. (2014). Intravital Two-Photon Imaging to Understand Bacterial Infections of the Mammalian Host. In: Vergunst, A., O'Callaghan, D. (eds) Host-Bacteria Interactions. Methods in Molecular Biology, vol 1197. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1261-2_5
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