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>
	<li>Saliba, W., Barnett-Griness, O., Rennert, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+obesity+and+urinary+tract+infection.">The association between obesity and urinary tract infection.</a> Eur J Intern Med. 24 (2), 27-31 (2012).</li><li>Semins, M., Shore, A., Makary, M., Weiner, J., Matlaga, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+obesity+on+urinary+tract+infection+risk.">The impact of obesity on urinary tract infection risk.</a> Urology. 79 (2), 266-269 (2011).</li><li>Zilberberg, M., Shorr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secular+trends+in+gram-negative+resistance+among+urinary+tract+infection+hospitalizations+in+the+United+States,+2000-2009.">Secular trends in gram-negative resistance among urinary tract infection hospitalizations in the United States, 2000-2009.</a> Infect Control Hosp Epidemiol. 34 (9), 940-946 (2013).</li><li>Whiting, D., Guariguata, L., Weil, C., Shaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IDF+diabetes+atlas:+global+estimates+of+the+prevalence+of+diabetes+for+2011+and+2030.">IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030.</a> Diabetes Res Clin Pract. 94 (3), 311-321 (2011).</li><li>Wild, S., Roglic, G., Green, A., Sicree, R., King, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+prevalence+of+diabetes:+estimates+for+the+year+2000+and+projections+for+2030.">Global prevalence of diabetes: estimates for the year 2000 and projections for 2030.</a> Diabetes Care. 27 (5), 1047-1053 (2004).</li><li>Ma, D., Gulani, V., Seiberlich, N., Liu, K., Sunshine, J., Duerk, J., 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=Magnetic+resonance+fingerprinting.">Magnetic resonance fingerprinting.</a> Nature. 495 (7440), 187-192 (2013).</li><li>Lu, L., Sedor, J., Gulani, V., Schelling, J., O'Brien, A., Flask, C. 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=Use+of+diffusion+tensor+MRI+to+identify+early+changes+in+diabetic+nephropathy.">Use of diffusion tensor MRI to identify early changes in diabetic nephropathy.</a> Am J Nephrol. 34 (5), 476-482 (2011).</li><li>Rosen, D., Hooton, T., Stamm, W., Humphrey, P., 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=Detection+of+intracellular+bacterial+communities+in+human+urinary+tract+infection.">Detection of intracellular bacterial communities in human urinary tract infection.</a> PLoS Med. 4 (12), e329 (2007).</li><li>Torine, L. 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. Med. Microbiol. 43 (1), 33-36 (1995).</li><li>Fernandes, P., Shipkowitz, N., Bower, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+for+studying+the+pathogenesis+and+treatment+of+pyelonephritis.">Murine models for studying the pathogenesis and treatment of pyelonephritis.</a> Adv. Exp. Med. Biol. 224, 35-51 (1987).</li><li>Kaye, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+water+diuresis+on+spread+of+bacteria+through+the+urinary+tract.">The effect of water diuresis on spread of bacteria through the urinary tract.</a> J. Infect. Dis. 124 (3), 297-305 (1971).</li><li>Fierer, J., Tainer, L., Braude, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteremia+in+the+pathogenesis+of+retrograde+E.+coli+pyelonephritis+in+the+rat.">Bacteremia in the pathogenesis of retrograde E. coli pyelonephritis in the rat.</a> Am. J. Pathol. 64 (2), 443-456 (1971).</li><li>Nickel, J., Olson, M., Costerton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+experimental+bacterial+prostatitis.">Rat model of experimental bacterial prostatitis.</a> Infection. 19 (3), S126-S130 (1991).</li><li>Hagberg, L., Engberg, I., Freter, R., Olling, S., Eden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascending,+unobstructed+urinary+tract+infection+in+mice+caused+by+pyelonephritogenic+Escherichia+coli+of+human+origin.">Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin.</a> Am Soc Microbiol. 40 (1), 273-283 (1983).</li><li>Kurosaka, Y., Ishida, Y., Yamamura, E., Takase, H., Otani, T., Kumon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-surgical+rat+model+of+foreign+body-associated+urinary+tract+infection+with+Pseudomonas+aeruginosa.">A non-surgical rat model of foreign body-associated urinary tract infection with Pseudomonas aeruginosa.</a> Microbiol. 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>
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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>
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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>
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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>
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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>
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		<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>
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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>
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		<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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			<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>
</table>

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?

intrarenal-injection-escherichia-coli-rat-model

Research

JoVE Journal

Medicine

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of intraoperative arrest in the field of cardiac surgery. With recent advances in cardiac surgery, the demand for prolonged VF studies under perfusion has increased. However, the field lacks simple, reliable, and reproducible animal models of chronic ventricular fibrillation. This protocol induces long-term VF through alternating current (AC) electrical stimulation of the epicardium. Different conditions were used to induce VF, including continuous stimulation with a low or high voltage to induce long-term VF and stimulation for 5 min with a low or high voltage to induce spontaneous long-term VF. The success rates of the different conditions, as well as the rates of myocardial injury and recovery of cardiac function, were compared. The results showed that continuous low-voltage stimulation induced long-term VF and that 5 min of low-voltage stimulation induced spontaneous long-term VF with mild myocardial injury and a high rate of recovery of cardiac function. However, the low-voltage, continuously stimulated long-term VF model had a higher success rate. High-voltage stimulation provided a higher rate of VF induction but showed a low defibrillation success rate, poor recovery of cardiac function, and severe myocardial injury. On the basis of these results, continuous low-voltage epicardial AC stimulation is recommended for its high success rate, stability, reliability, reproducibility, low impact on cardiac function, and mild myocardial injury.

This protocol presents a model of long-term ventricular fibrillation in rat hearts induced by continuous stimulation with low-voltage alternating current. This model has a high success rate, is stable, reliable, and reproducible, has a low impact on cardiac function, and causes only mild myocardial injury.

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...