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 intra-peritoneally.</li>
	<li>After 3-5 min of exposure to isoflurane, check that the animal is anesthetized and exhibits no response to toe pinch.</li>
	<li>Further sedate rat with an intra-peritoneal injection of a mix of xylazine and ketamine: 75 mg/kg ketamine/10 mg/kg xylazine. When performing intra-peritoneal injections, draw back the needle to ensure that portions of the intestine or other vital organs have not been punctured.</li>
	<li>Inject 2 mg/kg of bupivacaine subcutaneously at the site of incision to provide topical pain relief.</li>
</ol>
2. Preparation of Surgical Area
<ol>
	<li>Sterilize surgical instruments and supplies before they are used for surgery and lay out on the surgical pad for sterility. Most instruments and supplies can be autoclaved and re-used.</li>
	<li>Use sterile gloves for all surgical procedures.</li>
	<li>Use an electric razor to shave the fur off the animal&#39;s right side. Shave the animal from the bottom of the rib cage to the top of the hind leg providing a large hair-free area for the incision.</li>
	<li>Place the animal on a sterile surgical pad to isolate the disinfected area from surrounding areas.</li>
	<li>Scrub the skin with a disinfectant such as povidone iodine or betadine. Start the scrubbing at the center of the surgical site and move to the outside in a circular manner. Repeat at least three times with a new wipe of povidone iodine or betadine.</li>
	<li>Scrub the surgical site with 70% alcohol swabs until skin is clear, as iodine may be toxic if absorbed.</li>
</ol>
3. Surgical Procedure
<ol>
	<li>Maintain this procedure under aseptic conditions.</li>
	<li>Position the anesthetized animal on a warm heating bed in left lateral decubitus position with right flank facing upwards. 
	NOTE: Care must be taken to maintain the animal&#39;s core body temperature at 35-37 &#176;C to prevent hypothermia. This warming bed should also be sterilized as needed to maintain aseptic conditions.</li>
	<li>Feel for the rib cage, and make a small 2-3 cm right dorsal retroperitoneal incision using a sterile size 10 scalpel blade starting at the bottom of the rib cage.</li>
	<li>Place sterile gauze longitudinally along either side of the incision.</li>
	<li>Dissect away the subcutaneous tissue, fat, and muscles in order to visualize and access the abdominal cavity. Use curved-bladed Mayo scissors to allow deeper penetration into wound and cut thick tissues.</li>
	<li>Once the liver is clearly visible and accessible, use blunt forceps to retract the liver upwards.</li>
	<li>Using another pair of blunt forceps in the other hand, expose the right kidney so it sits just outside of the abdominal cavity.</li>
	<li>Use the pointer finger and thumb of the left hand to hold the kidney in position. With the right hand, slowly and steadily inject 0.1 ml UTI89 E. coli solution (concentration between 1 x 108-1 x 109) from a sterile syringe into the renal pelvis (which appears as a white bubble)15. 
	NOTE: Prepare the bacterial titer as described in reference15.</li>
	<li>Place a strip of absorbable hemostat over the needle to prevent outflow of inoculum into the peritoneum. Slowly pull the needle out of the renal pelvis. 
	NOTE: Care should be taken not to pierce or infect surrounding tissues resulting in off-target infections and/or complications.</li>
	<li>Use a larger syringe to thoroughly rinse the kidney with normal saline before placing it back into the abdominal cavity.</li>
</ol>
4. Sutures
Note: Suture that will be buried in tissues should be 4-0 non-absorbable braided sutures. Absorbable or monofilament&#160;sutures may be used for body surfaces.
<ol>
	<li>Place the sutures evenly and as close to the tissue edge as possible to prevent obstruction of blood flow; typically no more than 0.3 cm from the edge is necessary.</li>
	<li>Grasp the skin and evert it slightly using a pair of fine toothed forceps, and rotate the needle holder into a pronated position in preparation for piercing the skin.</li>
	<li>Drive the needle through the full thickness of skin by supinating the wrist to rotate the needle and pass it through the skin.</li>
	<li>Repeat this process for the skin edge closest to the individual performing the procedure.</li>
	<li>Tighten the sutures enough to oppose the tissue edges. Any tighter will obstruct blood supply, slow wound healing and may result in dehiscence.</li>
	<li>Tie off the suture using square knots, as if it were a simple interrupted stitch, except that only the short strand is cut, leaving about a 3-4 mm tail.</li>
	<li>After the first stitch has been tied off, prepare to place a second stitch about 3 mm away from the first, and continue the running suture.</li>
	<li>Once the end of the incision is reached, do not pull the last stitch completely through. Instead, use the loop which is being held with the needle holder here as the short strand in order to tie off the distal end of the suture closure.</li>
	<li>Using instrument ties, tie off the suture using square knots. This results in 3 strands sticking up from the completed knot at the distal end.</li>
</ol>
5. Animal Recovery
<ol>
	<li>Inject 2.1 mg/kg Yohimbine intra-peritoneally after surgery to reverse anesthesia. Animals are expected to fully recover from the surgical procedure after 3-5&#160;hr.</li>
	<li>Keep the animal on a heating pad (to avoid hypothermia) and absorbent soft bedding after surgery.</li>
	<li>Provide rehydration with oral or parenteral fluids until it can return to normal feeding (within 24 hr). Inject approximately 0.6 ml of normal saline solution peritoneally immediately following surgery to limit dehydration effects.</li>
	<li>Provide analgesia as described in the IACUC approved Animal Care and Use Protocol. Inject 5 mg/kg carprofen subcutaneously for pain management.</li>
	<li>Monitor incisions regularly for swelling, exudate, pain, or dehiscence.</li>
</ol>
6. Validation via Magnetic Resonance Imaging
<ol>
	<li>Conduct in vivo MRI experiments on high field small animal MRI scanners.</li>
	<li>Induce anesthesia with 3% isoflurane in oxygen and position the animal with the right kidney at isocenter in the MRI scanner and appropriate radiofrequency coil. Provide animals with 1-2% isoflurane anesthesia continuously throughout the imaging procedure via a nosecone. 
	NOTE: For the in vivo MRI images shown in Figure 1, a rat-sized volume coil (inner diameter = 72 mm) was utilized.</li>
	<li>Use an animal monitoring and control system to maintain each animal&#39;s respiration rate (40-60 breaths/min) and core body temperature (35 &#177; 1 &#176;C).</li>
	<li>Use a multi-slice, multi-echo spin echo MRI acquisition to obtain high resolution, axial T2-weighted images of both the infected and control kidneys. Typical MRI acquisition parameters are repetition time = 5,000 msec, echo time = 40 msec, slice thickness = 2.0 mm, in-plane spatial resolution = 200 &#181;m, 3 signal averages, and an acquisition time of 8 min.</li>
</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. 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. 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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>

The authors have no other disclosures.

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).

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latex Surgical Gloves</td>
			<td>Henry Schein Animal Health</td>
			<td>21540</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>050033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td>Henry Schein Animal Health</td>
			<td>33197</td>
			<td>Inject IP</td>
		</tr>
		<tr height="21">
			<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...

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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 ...

intrarenal-injection-escherichia-coli-rat-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.9K Views.  Case Western Reserve University. 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.

Video: Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

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.

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 ...

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

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Chapters in this video

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

A Model of Chronic Nutrient Infusion in the Rat

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Acupuncture in a Rat Model of Asthma

Retinal Pathophysiological Evaluation in a Rat Model

Development of a Uterosacral Ligament Suspension Rat Model

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