Name: intrarenal injection of escherichia coli in a rat model of pyelonephritis
Uploaded: 2017-07-18T12:30:00
Description: Watch this Scientific Journal Video about Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis at JoVE.com

The authors would like to acknowledge the support of NIH/NIDDK K12 DK100014 (Lan Lu), the Case Comprehensive Cancer Center (NIH/NCI P30 CA43703), and the Clinical and Translation Science Collaborative of Cleveland (NIH/NCATS UL1 TR000439).

All animal studies were performed according to approved Institutional Animal Care and Use Committee (IACUC) protocols at Case Western Reserve University. The duration of the surgical procedure described below is approximately 45-60 min. The MRI procedure itself is approximately 15 min for each time point.
1. Anesthesia
<ol>
	<li>Anesthetize rat in isoflurane chamber set at 2% isoflurane mixed with oxygen to facilitate animal handling and restraint prior to administering injectable anesthesia...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+tract+infection:+diabetic+women's+strategies+for+prevention.">Urinary tract infection: diabetic women's strategies for prevention.</a> Br J Nurs. 20 (13), 791-792 (2011).</li><li>Rosen, D., Hung, C., Kline, K., Hultgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptozocin-induced+diabetic+mouse+model+of+urinary+tract+infection.">Streptozocin-induced diabetic mouse model of urinary tract infection.</a> Infect Immun. 76 (9), 4290-4298 (2008).</li><li>Larsson, P., Kaijser, B., Mattsby-Baltzer, I., Olling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+ascending+acute+pyelonephritis+caused+by+Escherichia+coli+or+proteus+in+rats.">An experimental model for ascending acute pyelonephritis caused by Escherichia coli or proteus in rats.</a> J Clin Pathol. 33 (4), 408-412 (1980).</li><li>Gupta, R., Ganguly, N., Ahuja, V., Joshi, K., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ascending+non-obstructive+model+for+chronic+pyelonephritis+in+BALB/c+mice.">An ascending non-obstructive model for chronic pyelonephritis in BALB/c mice.</a> J. 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Immunol. 45 (1), 9-15 (2001).</li><li>Anderson, B., Jackson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyelitis,+an+important+factor+in+the+pathogenesis+of+retrograde+pyelonephritis.">Pyelitis, an important factor in the pathogenesis of retrograde pyelonephritis.</a> J Exp Med. 114 (3), 375-384 (1961).</li><li>Anderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesico-ureteric+reflux.">Vesico-ureteric reflux.</a> J R Soc Med. 55 (6), 419-426 (1962).</li></ol>

Ascending acute pyelonephritis in rodents (i.e. mice and rats) can be produced by transurethral catheterization.16,17,18 This transurethral infection method is advantageous in that it is non-invasive and mimics the human pathophysiology of ascending infection.17,18,19,20 However, this method c...

Rodent models have been used to study numerous human disease manifestations, including pyelonephritis and urinary tract infections (UTI). UTIs are a global health problem, and can impact children, men, and women of all ages.1,2,3 The initial manifestation of UTIs includes cystitis, and if the infection ascends along the ureter, a kidney infection (pyelonephritis) may follow. At the same time, the prevalence of diabetes is approaching 400 million people worldwide.4,5 Importantly, UTI incidence may be up to 4 times higher in patients who are obese or have type 2 diabetes mellitus, resulting in increased risk of recurrent UTI infection (rUTI), sepsis, renal fibrosis from pyelonephritis, and bladder dysfunction.6,7,8 Rodent models are important in studying UTIs, because current antibiotic therapies produce a sustained, preventative response only in a subset of UTI patients. To improve clinical UTI care, the key steps are to understand the mechanism of rUTI and its pathophysiological processes from acute infection to inflammation to fibrosis, as well as the impact of type 2 diabetes mellitus.
The goal of improving animal models is to develop techniques that allow for a more accurate evaluation of disease progression and therapeutic interventions. Several different approaches have been employed to induce pyelonephritis in rats and/or mice to study the pathophysiology of kidney damage, the effect of antibiotic treatment, and other aspects of the natural course of UTIs. A common approach to establish retrograde UTI is transurethral catheterization.10,11,12,13 This method introduces bacteria via the urethra into the urinary bladder of anesthetized animals. While this technique closely simulates human pyelonephritis, the actual incidence and magnitude of pyelonephritis infection can be highly variable because of multiple factors including a lack of spontaneous ureteric reflux or urinary voiding during or immediately following inoculation.11 As a result, the experimental variability in inducing an ascending pyelonephritis infection can limit the utility of this model to study kidney infections as well as therapeutic strategies.
This report describes a surgical pyelonephritis rat model where E. coli is directly injected into the rat kidney. Despite this rat model being invasive, the amount of E. coli delivered to the kidney can be effectively controlled enabling a robust kidney infection and inflammation.14 Within this procedure, we also describe how these induced renal infections can be monitored longitudinally with in vivo Magnetic Resonance Imaging (MRI).

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			<td height="21" style="height:21px;width:216px;">Absorbing Pad</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:168px;">14-127-47</td>
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			<td height="21" style="height:21px;width:216px;">Sterile Cotton Gauze Pad</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:168px;">22-415-469</td>
			<td></td>
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			<td height="21" style="height:21px;">Latex Surgical Gloves</td>
			<td>Henry Schein Animal Health</td>
			<td>21540</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Curved Mayo Scissors</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:168px;">S17341</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Straight Blunt Foreceps</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:168px;">08-895</td>
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			<td height="21" style="height:21px;width:216px;">Scalpel Handle&#160;</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:168px;">08-913-5</td>
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			<td height="21" style="height:21px;width:216px;">Sterile Scalpel Blades</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:168px;">53220</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">1mL Luer-Lok Syringe</td>
			<td style="width:195px;">BD Biosciences</td>
			<td>309628</td>
			<td>For bacterial injections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20mL Luer-Lok Syringe&#160;</td>
			<td style="width:195px;">BD Biosciences</td>
			<td>301031</td>
			<td>For saline wash</td>
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			<td height="21" style="height:21px;width:216px;">Hemostat</td>
			<td style="width:195px;">Seneca Medical</td>
			<td style="width:168px;">240267</td>
			<td></td>
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			<td height="21" style="height:21px;">23 G 3/4 in. Needle&#160;</td>
			<td style="width:195px;">BD Biosciences</td>
			<td style="width:168px;">305143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30 G 1 in. Needle&#160;</td>
			<td style="width:195px;">BD Biosciences</td>
			<td style="width:168px;">305128</td>
			<td></td>
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			<td height="21" style="height:21px;">U-100 Insulin Syringe</td>
			<td>Exel International&#160;</td>
			<td style="width:168px;">25846</td>
			<td>For medication injections</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>050033</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Xylazine</td>
			<td>Henry Schein Animal Health</td>
			<td>33197</td>
			<td>Inject IP</td>
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			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td>Patterson Vetrinary&#160;</td>
			<td>07-881-9413</td>
			<td>Inject IP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yohimbine (Atipamezole)</td>
			<td style="width:195px;">Patterson Vetrinary&#160;</td>
			<td>07-867-7097</td>
			<td>Inject IP after surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bupivacaine (Marcaine)</td>
			<td style="width:195px;">Patterson Vetrinary&#160;</td>
			<td>07-890-4584</td>
			<td>Inject SQ at site of incision&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofen (Rimadyl)</td>
			<td style="width:195px;">Patterson Vetrinary&#160;</td>
			<td>07-844-7425</td>
			<td>Should be kept at 4 &#7506;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 Chromic Gut Suture</td>
			<td style="width:195px;">Ethicon Inc.</td>
			<td>U203H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Braided Vicryl Suture</td>
			<td>Ethicon Inc.</td>
			<td>J304H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL SubQ Syringe</td>
			<td>BD Biosciences</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">E. coli&#160; UTI89 or CFT073</td>
			<td style="width:195px;">ATCC</td>
			<td style="width:168px;">700928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgicel Absorbable Hemostat</td>
			<td style="width:195px;">Ethicon Inc.</td>
			<td>ETH1951CS&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biospec 9.4T MRI&#160;</td>
			<td style="width:195px;">Bruker&#160;</td>
			<td style="width:168px;">94/20 USR</td>
			<td></td>
		</tr>
	</tbody>
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intrarenal injection of escherichia coli in a rat model of pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Medical imaging techniques offer the opportunity to non-invasively assess UTI and therapeutic efficacy. Therefore, MRI was utilized to validate induction of acute infection after injection of 1-2 x 107 UTI89 E. coli, and to visualize the changes in the kidney before and after surgery. Figure 1a-b shows a progressively increasing region of kidney infection (yellow arrows). MRI images obtained for each animal at days 1 and 4 post-infection help character...

Watch this Scientific Journal Video about Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis at JoVE.com

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

The overall goal of this surgical rat model of Peylonephritis is to provide a method that does not require renal reflux, which is used in other models. This method can help answer key questions in the the medical field such as how best to administer antibiotic therapy, how we can monitor the effects of recurrent infections or residual infections after therapy that can lead to recurrence. The main advantage of this technique is that this model directly applies bacteria to the kidney in a reasonably reliable quantity without the requirement of renal reflux, which can add some variability in terms of infection.Prior to starting this procedure, prepare the bacterial injection bolus containing between one million and 10 million cells. After anesthetizing the rat and preparing its skin for surgery, apply vet eye ointment to the eyes to prevent dryness. Position the rat on a warmed surgical platform in a left decubitus position with its right flank facing up.Scrub the skin with a disinfectant. Repeat three times and then scrub the surgical site with 70%alcohol swap until the skin is clear. To begin the surgery, using a Number 10 scalpel, make a small, two to three centimeter right dorsal retroperitoneal incision beginning at the bottom of the rib cage.Continue by dissecting away the subcutaneous tissue, fat and muscles to ultimately visualize and access the abdominal cavity. Once the liver is clearly visible and accessible, use blunted forceps to retract it upwards. Then, using a second pair of blunted forceps, expose the right kidney so it sits just outside of the abdominal cavity.Now, position the kidney for injection in the nondominant hand. Using the dominant hand, precisely target the injection needle on the renal pelvis. Then, slowly and steadily inject 0.1 milliliters of bacterial or saline solution.Here, saline is being injected. The bolus will look like a white bubble in the tissue. Before retracting the needle, place a strip of absorbable hemostat over the injection site to prevent an outflow of the inoculum into the peritoneum.Then, slowly pull the needle out. Now, using a large syringe, thoroughly rinse the kidney with normal saline. Then, return the kidney to the abdominal cavity.For the surgical site closure, use absorbable or monofilament sutures in body tissue and 4-0 nonabsorbable braided sutures on body surfaces. To place sutures, start at the site furthest from the operator, using fine toothed forceps to grasp the skin and avert it slightly. Then, rotate the needle holder into a pronated position and supinate the wrist to rotate the needle and drive it through the full thickness of the skin.Tighten the suture just enough to expose the tissue edges. Any tighter will obstruct the blood supply and thus slow wound healing and possibly result in dehiscence. Then, tie off the suture using a square knot, as if it were a simple interrupted stitch, except that only the short strand is cut, leaving a three to four millimeter tail.Now, placing each subsequent stitch about three millimeters from the previous, continue the running suture. Once the end of the incision is reached, do not pull the last stitch completely through. Instead, use the loop being held by the needle holder as the short strand and tie off the distal end of the suture closure.Now, using instrument ties, tie off the suture using square knots. The result will be three strands sticking up from a completed knot. After providing analgesics and a 0.6 milliliter peritoneal injection of normal saline, observe the animal while it recovers from the anesthesia on a heating pad with absorbent soft bedding.Over the first 24 hours of recovery, rehydrate the animal with oral or parenteral fluids until it can return to normal feeding. Thereafter, regularly monitor the incision for swelling, exudate or dehiscence and provide analgesics for pain as needed. An acute peylonephritis infection was induced by injection of one million and 10 million UT189 E.coli cells into the rodent model.MRI of the kidney showed a progressive increase in the region of infection from one to four days after injection. Once mastered, this technique can be done in 30 minutes if performed properly. While attempting this procedure, it's important to remember to check on the animals following surgery to watch for signs of E.Coli infection outside of the kidney, especially at the incision points, to make sure that the animals do not become overly septic.Following this procedure, other methods, like MRI scanning, can be performed in order to answer additional questions like, is the infection viable, how fast is the infection growing, and is this therapy effective?

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