This research was supported by grant 1K01DK079019A2 (P.T.) from the NIH/NIDDK.

1. Bacteria Preparation for Mouse Inoculation
The following must be done under aseptic conditions.
<ol>
	<li>Take one 17 x 100 mm polypropylene tubes with caps and add 3 ml of fresh Luria broth (LB) media. Using autoclaved tips, take some frozen bacteria glycerol stock of strain CP12 and transfer a small quantity of the culture into the tube containing the LB media. Replace the tube cap so that oxygen is still able to enter the tube - the culture needs to grow under aerobic conditions. Place tube at 37 &deg;C in a shaking incubator at 220 rpm overnight.</li>
	<li>The next day, transfer 5 &mu;l of overnight culture to a fresh tube with 3 ml LB media and grow under static conditions in an incubator overnight at 37 &deg;C.</li>
	<li>On day 3 transfer 40 &mu;l of culture into a a 50 ml tube containing 40 ml LB media each. Place in a 37 &deg;C static incubator overnight.</li>
	<li>Turn on and set centrifuge to 4 &deg;C. Once centrifuge has reached final temperature, transfer each 40 ml culture into a 40 ml Nalgene centrifuge tube.</li>
	<li>Weigh tubes to balance centrifuge - adjust volume with LB media as needed. Spin tubes at 6,000 rpm for 20 min.</li>
	<li>Carefully remove supernatant and gently suspend bacterial pellet in 40 ml sterile PBS.</li>
	<li>Repeat step 1.5 with PBS.</li>
	<li>Aspirate off supernatant and suspend bacteria in 500 &mu;l sterile PBS. Transfer the solution into a 1.5 ml microfuge tube. Take 10 &mu;l of suspension and dilute in 990 &mu;l of ice-cold PBS. Use a spectrophotometer to read the OD420 nm to determine the volume needed for inoculation. To determine the appropriate volume of ice-cold PBS to suspend pellet in: Take the OD420 nm number (in ml) and subtract the estimated volume of the bacterial pellet [ex. OD420 nm = 0.545, pellet approximately 0.075 ml. 0.545 - 0.075 = 0.470].</li>
	<li>Remove 10 &mu;l of bacterial suspension and dilute in 990 &mu;l ice-cold PBS. Read the OD420 nm. The goal is to achieve an OD420 nm value for the diluted suspension of 1.000 &plusmn; .010. If the number of your dilution is above this target, add more volume to the suspension and take another reading. If the number is below the .990 then spin down the suspension and repeat OD420 nm reading until obtaining desired reading.</li>
</ol>
2. Animal Infection
Mice (5 to 7 weeks old, ten per group) were purchased from Jackson Laboratory (Bar Harbor, ME).
<ol>
	<li>Mice are anesthetized by inhalation of 1-4% isoflurane in a Plexiglas chamber and are monitored until recumbent.</li>
	<li>One mouse at a time is taken for instillation of bacteria by catheterization with a polyethylene (PE10) catheter attached to a modified 30 G needle of a glass Hamilton syringe (length of 1.5-2.0 cm). The catheter is inserted to the hub of the needle. The mouse is placed on its dorsal surface under anesthesia maintained using a nose cone.</li>
	<li>The penis of the mouse is extruded by gentle pressure and liberal amounts of lubricant on cotton swabs is used for lubrication of the entrance to the penile urethra.</li>
	<li>10 &mu;l of phosphate-buffered saline containing 1 x 108 bacteria is introduced into the urethra of anesthetized mice following catheterization.</li>
	<li>Mice are maintained in an anesthetized state for 15 min in the Plexiglas chamber after bacterial introduction to allow bacterial attachment and to prevent immediate urination.</li>
	<li>Mice are placed back in their cages and monitored for the next 24 hr.</li>
</ol>
3. Behavior Testing
Mice were tested prior to infection (baseline) and on days 3, 7, 14, 21 and 28 after infection. Referred hyperalgesia and tactile allodynia was tested using von Frey filaments applied to the abdomen11,12 and the plantar region of the hind paw13. Testing was perfomed at a fixed time-of-day, standard methodology and single experimenter testing of all animals were employed. Blinded testing of groups was utilized to combat the limitations of behavior-based pain testing in animal models. Five individual von Frey filaments with forces of 0.04, 0.16, 0.4, 1.0 and 4.0 g (Stoelting, USA) were applied to the abdomen and frequency of withdrawal responses was calculated.
<ol>
	<li>After a 30 min period of acclimatization, mice are placed in individual Plexiglas chambers (6 x 10 x12 cm) made in-house with a stainless steel wire grid floor.</li>
	<li>Referred hyperalgesia and tactile allodynia were tested using the five von Frey filaments. Each filament was applied for 1-2 sec with an inter-stimulus interval of 5 sec for a total 10 times, and the hairs were tested in ascending order of force.</li>
	<li>Each filament is applied to the lower abdominal area in the general vicinity of the prostate and care was taken to stimulate different areas within this region to avoid desensitization or "wind up" effects. Filaments were applied in increasing order of force for 1-2 sec with at least 5 sec intervals between stimulations for a total of 10 times.</li>
	<li>Three types of behavior are considered a positive response to filament stimulation: 1) sharp retraction of the abdomen; 2) immediate licking or scratching of the area of filament stimulation; 3) jumping.</li>
	<li>Response frequency was calculated as the percentage of positive response (out of 10, e.g. 5 responses of 10 = 50%) and data was reported as the mean percentage of response frequency &plusmn; SE</li>
	<li>Animals with more than 25 positive total baseline responses are excluded from the study.</li>
	<li>Tactile allodynia was tested on the plantar region of the hind paw using von Frey filaments with forces of 0.04, 0.16, 0.4, 1.0 and 4.0 g. The median 50% withdrawal threshold (5) was assessed using the up-down method where testing was started with 0.04 g filament applied perpendicularly to the plantar surface of the hind paw until the filament bent slightly. Filaments were tested in ascending order until a positive response was observed. A positive response to the filament was defined as either a sharp withdrawal of the paw or licking of the test paw. When a positive response was recorded the next weaker filament was applied, and if a negative response was observed, then the next stronger filament was applied.</li>
	<li>The experiments and methodology described have been reviewed and approved by the Northwestern University animal care and use committee.</li>
</ol>

<ol>
	<li>Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+uropathogenic+Escherichia+coli.">Molecular epidemiology of uropathogenic Escherichia coli.</a> Journal of infection and chemotherapy official journal of the Japan Society of Chemotherapy. 13, 68-73 (2007).</li><li>Rudick, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uropathogenic+Escherichia+coli+induces+chronic+pelvic+pain.">Uropathogenic Escherichia coli induces chronic pelvic pain.</a> Infect. Immun. 79, 628-635 (2011).</li><li>Pontari, M. A., Ruggieri, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+in+prostatitis/chronic+pelvic+pain+syndrome.">Mechanisms in prostatitis/chronic pelvic pain syndrome.</a> The Journal of urology. 172, 839-845 (2004).</li><li>Schaeffer, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Chronic+prostatitis+and+the+chronic+pelvic+pain+syndrome.">Clinical practice. Chronic prostatitis and the chronic pelvic pain syndrome.</a> The New England journal of medicine. 355, 1690-1698 (2006).</li><li>Rudick, C. N., Schaeffer, A. J., Thumbikat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+prostatitis+induces+chronic+pelvic+pain.+American+journal+of+physiology.">Experimental autoimmune prostatitis induces chronic pelvic pain. American journal of physiology.</a> Regulatory, integrative and comparative physiology. 294, R1268-R1275 (2008).</li><li>Elkahwaji, J. E., Ott, C. J., Janda, L. M., Hopkins, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+for+acute+bacterial+prostatitis+in+genetically+distinct+inbred+strains.">Mouse model for acute bacterial prostatitis in genetically distinct inbred strains.</a> Urology. 66, 883-887 (2005).</li><li>Hultgren, S. J., Porter, T. N., Schaeffer, A. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+type+1+pili+and+effects+of+phase+variation+on+lower+urinary+tract+infections+produced+by+Escherichia+coli.">Role of type 1 pili and effects of phase variation on lower urinary tract infections produced by Escherichia coli.</a> Infect. Immun. 50, 370-377 (1985).</li><li>Jarrell, J., Giamberardino, M. A., Robert, M., Nasr-Esfahani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bedside+testing+for+chronic+pelvic+pain:+discriminating+visceral+from+somatic+pain.">Bedside testing for chronic pelvic pain: discriminating visceral from somatic pain.</a> Pain research and treatment. 2011, 692102 (2011).</li><li>Jarrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+Cutaneous+Allodynia+in+Association+with+Chronic+Pelvic+Pain.">Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain.</a> J. Vis. Exp. (28), e1232 (2009).</li><li>Giamberardino, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscero-visceral+hyperalgesia:+characterization+in+different+clinical+models.">Viscero-visceral hyperalgesia: characterization in different clinical models.</a> Pain. 151, 307-322 (2010).</li><li>Laird, J. M., Martinez-Caro, L., Garcia-Nicas, E., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+visceral+pain+and+referred+hyperalgesia+in+the+mouse.">A new model of visceral pain and referred hyperalgesia in the mouse.</a> Pain. 92, 335-342 (2001).</li><li>Laird, J. M., Souslova, V., Wood, J. N., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+visceral+pain+and+referred+hyperalgesia+in+Nav1.8+(SNS/PN3)-null+mice.">Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice.</a> J. Neurosci. 22, 8352-8356 (2002).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of neuroscience. 53, 55-63 (1994).</li><li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+type+1+pilus+expression+in+Escherichia+coli+to+ascending+urinary+tract+infections+in+mice.">Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.</a> Infect. Immun. 55, 373-380 (1987).</li><li>Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+bacterial+inflammation+of+the+mouse+prostate.">Acute bacterial inflammation of the mouse prostate.</a> The Prostate. 72, 307-317 (2012).</li><li>Rivero, V. E., Cailleau, C., Depiante-Depaoli, M., Riera, C. M., Carnaud, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-obese+diabetic+(NOD)+mice+are+genetically+susceptible+to+experimental+autoimmune+prostatitis+(EAP).">Non-obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP).</a> Journal of autoimmunity. 11, 603-610 (1998).</li><li>Pakarainen, T., Zhang, F. P., Makela, S., Poutanen, M., Huhtaniemi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone+replacement+therapy+induces+spermatogenesis+and+partially+restores+fertility+in+luteinizing+hormone+receptor+knockout+mice.">Testosterone replacement therapy induces spermatogenesis and partially restores fertility in luteinizing hormone receptor knockout mice.</a> Endocrinology. 146, 596-606 (2005).</li><li>Taguchi, O., Kojima, A., Nishizuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+prostatitis+after+neonatal+thymectomy+in+the+mouse.">Experimental autoimmune prostatitis after neonatal thymectomy in the mouse.</a> Clinical and experimental immunology. 60, 123-129 (1985).</li></ol>

No conflicts of interest declared.

Infection of the mouse prostate with UPEC allows for the in vivo modeling of events that may be involved in the pathogenesis of bacterial prostatitis, CP/CPPS or as a predisposing event in chronic inflammation. The methods described for bacterial preparation and instillation draw upon a large body of literature on UPEC models in female urinary tract infection7,14. The model has wide applicability for studying pathogenesis, testing potential vaccine candidates and mechanisms of immune modulation. The a...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Culture tubes</td>
 <td>BD Falcon</td>
 <td>352059</td>
 </tr>
 <tr>
 <td>LB Broth Miller</td>
 <td>EMD</td>
 <td>EM1.10285.0500 </td>
 </tr>
 <tr>
 <td>Nalgene Centrifuge Tubes</td>
 <td>Thermo</td>
 <td>3118-0050</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Butler Schein</td>
 <td>NDC 11695-6776-1</td>
 </tr>
 <tr>
 <td>Catheter needle 30G</td>
 <td>Hamilton</td>
 <td>91030</td>
 </tr>
 <tr>
 <td>PE10 tubing</td>
 <td>BD intramedic</td>
 <td>427400</td>
 </tr>
 <tr>
 <td>Von Frey Filaments</td>
 <td>Stoelting</td>
 <td>58025-31</td>
 </tr>
 </tbody>
</table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

We examined NOD/ShiLtj and C57BL/6J male mice for the presence of chronic pain upon bacterial infection. Male mice (5 to 7 weeks old) were instilled with saline or bacteria into the urethra (Figure 1). Mechanical stimulation of the pelvic area with von Frey filaments of saline-instilled C57BL/6J mice and UPEC-infected C57BL/6J mice resulted in a response frequency that did not change during the 28-day course of the experiment (Figure 1). In contrast, UPEC-infected NOD mice exhibited resp...

Watch this Scientific Journal Video about Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis at JoVE.com

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

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Immunology and Infection

15.7K 次观看.  Northwestern University Feinberg School of Medicine. 在调解盆腔疼痛慢性前列腺炎前列腺感染可能是一个促进因素。我们的程序编制标准化的细菌接种的细菌进入尿道的雄性小鼠和方法测量触觉异常的小鼠，随着时间的推移，滴入。

视频: Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...