The authors would like to acknowledge the receipt of both the Pediatric Orthopaedic Society of North America Biomet Spine Grant and the National Institutes of Health Clinical and Translational Science Institute KL2 Grant, and the HH Lee Surgical Research Grant as major funding sources for these experiments.

All animals were handled in strict accordance with good animal practice as defined in the federal regulations as set forth in the Animal Welfare Act (AWA), the 1996 Guide for the Care and Use of Laboratory Animals, PHS Policy for the Humane Care and Use of Laboratory Animals, as well as the institution&#8217;s policies and procedures as set forth in the Animal Care and Use Training Manual, and all animal work was approved by the University of California Los Angeles Chancellor&#8217;s Animal Research Committee (ARC).
1. S. aureus bioluminescent strain choice
<ol>
	<li>Use the bioluminescent S. aureus strain Xen36 as the inoculum of interest. 
	NOTE: This strain was derived from the parental strain S. aureus ATCC-49525, which is a clinical isolate from a septic patient. S. aureus Xen36 uniquely utilizes a luxABCDE operon, which is optimized and integrated into the host&#8217;s native plasmid.19 As a result, the Xen36 strain is capable of producing a blue-green bioluminescent light with a peak wavelength emission of 490 nm. This emission signal is only produced by living metabolically active bacterial organisms.</li>
</ol>
2. Preparation of S. aureus for inoculation
<ol>
	<li>Add 200 &#956;g/mL kanamycin to Luria Broth plus 1.5% agar to isolate S. aureus Xen36 from potential contaminants, utilizing the kanamycin resistance gene linked to the lux operon19.</li>
	<li>Streak S. aureus Xen36 bacteria onto tryptic soy agar plates (tryptic soy broth [TSB] plus 1.5% agar) and incubate at 37 &#176;C for 12-16 h.</li>
	<li>Isolate single colonies of S. aureus Xen36 and individually culture in TSB for 12-16 h at 37 &#176;C in a shaking incubator (200 rpm).</li>
	<li>Dilute resultant culture at a 1:50 ratio.</li>
	<li>Culture for additional 2 h at 37 &#176;C to isolate midlogarithmic phase bacteria.</li>
	<li>Pellet, resuspend, and wash bacteria in phosphate buffered saline (PBS) three times.</li>
	<li>Measure the absorbance at 600 nm. The ideal OD600 is between 0.700 and 0.750 which corresponds to 4x105 CFU/mL. Perform serial dilutions to achieve desired bacterial inoculum (1 x 103&#160; CFU/2 &#181;L). 
	NOTE: The optimal concentration of Xen36 for the establishment of a chronic infection was found to be 1 x 103 CFU. Lower dosing of bacteria was cleared by the host immune system and higher dosing caused wound breakdown. Wound breakdown does not differentiate between a deep implant infection and a superficial wound infection and is therefore avoided in this model (Figure 1)20.</li>
</ol>
3. Mice
<ol>
	<li>Use 12-week old male C57BL/6J wild-type mice.</li>
	<li>House mice in cages with a maximum of 4 at a time.</li>
	<li>Keep water available at all times. Maintain a 12-hour light/dark cycle and do not perform experimentation during the dark phase of the cycle.</li>
	<li>Use alfalfa-free chow for feeding due to potential interference with fluorescent signaling.</li>
	<li>Have research or veterinary staff assess mice daily to ensure the well-being of the animals throughout the entirety of the experiment.</li>
</ol>
4. Mouse surgical procedures
<ol>
	<li>Induce anesthesia by placing mice in an isoflurane (2%) chamber for approximately 5 minutes. Confirm appropriate depth of anesthesia by monitoring respirations to remain rhythmic and slower than when awake and not changing in response to noxious stimuli (e.g., surgical manipulation, toe pinch).</li>
	<li>Transfer anesthetized mice to a preparation station and remove hair from the sacrum to the upper thoracic spine with rodent clippers.</li>
	<li>Clean and sterilize the skin with triple washes of alternating betadine solution and isopropyl alcohol.</li>
	<li>Transfer anesthetized and sterilized mice in the prone position to a sterile surgical bed maintaining anesthesia with administration of inhaled isoflurane (2%) via nose cone.</li>
	<li>Maximally flex the hips and identify the position of the knee at the level of the spine to approximate the lumbar 4 vertebral body.</li>
	<li>Make a longitudinal 2 cm incision through skin with a 15-blade surgical scalpel.</li>
	<li>Palpate the spinous processes to confirm midline and continue the incision down to bone.</li>
	<li>Dissect subperiosteally on the right side of the L4 spinous process, extending laterally to the transverse process.</li>
	<li>Pass an absorbable braided suture size 5-0 cephalad and caudad to the L4 body through the fascia and leave open, in preparation for future closure.</li>
	<li>Using a 25 G spinal needle, ream the spinous process of L4 using a 25 G spinal needle and insert a 0.1 mm diameter, 1 cm long &#8220;L-shaped&#8221; surgical grade stainless-steel implant along the lamina with the long arm laying cephalad.</li>
	<li>Inoculate the implant with 1 x 103 CFUs/2 &#181;L bioluminescent S. aureus Xen36, taking care to ensure all solution contacts the implant.</li>
	<li>Wait approximately 10 seconds before tying the previously passed absorbable suture following inoculation to ensure containment of inoculum on the implant.</li>
	<li>Close skin in running fashion with absorbable suture.</li>
	<li>Administer pain medicine via subcutaneous injection of buprenorphine (0.1 mg/kg) immediately postop and then every 12 hours for 3 days thereafter.</li>
	<li>Recover mice on a heating pad and monitor for return to normal activity.</li>
	<li>Obtain postoperative radiographs to confirm appropriate placement of implant.</li>
</ol>
5. Longitudinal In vivo bioluminescence imaging to measure bacterial burden
<ol>
	<li>Anesthetize mice with inhaled isoflurane (2%). Confirm appropriate depth of anesthesia by monitoring respirations to remain rhythmic and slower than when awake and not changing in response to noxious stimuli (e.g., surgical manipulation, toe pinch).</li>
	<li>Remove hair from the sacrum to the upper thoracic spine with rodent clippers.</li>
	<li>Load mice onto field of view of bioluminescent imaging platform to perform in vivo bioluminescence imaging (BLI)19.</li>
	<li>Capture bioluminescent signal over a 5 min acquisition time. Utilize large binning settings with a 15 cm field of view (B).</li>
	<li>Repeat steps 5.1-5.4 on postoperative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35 (or other days based on specific experimental design) to monitor bacterial burden.</li>
	<li>Present BLI data via color scale and overlay on a grayscale photograph. Isolate a standard ovoid region of interest (ROI) using BLI software to quantify BLI in total flux (photons per second) or mean maximum flux (photons/second/centimeter2/steradian).</li>
</ol>
6. Quantify bacteria adherent to implants and surrounding tissue
<ol>
	<li>Euthanize mice on POD 35 or an alternative post-operative date of choice with exposure to carbon dioxide in accordance with AVMA Guidelines. Confirm euthanasia with secondary cervical dislocation.</li>
	<li>Sterilize the dorsal skin according to Step 4.3 and position the mouse prone on a sterile surgical field.</li>
	<li>Sharply incise the previous incision using a 15-blade surgical scalpel.</li>
	<li>Use sterile scissors to bluntly dissect to the L4 spinous process and identify the surgical implant.</li>
	<li>Use a needle driver to gently twist and remove the implant from its position in the L4 spinous process.</li>
	<li>Using sterile forceps and scissors, harvest approximately 0.1 g of spinous process bone and soft tissue immediately surrounding the surgical implant and place in 1 mL of PBS in small conical rhino tube with 4 sharp homogenizing beads.</li>
	<li>Record weight of soft tissue by weighing conical tube before and after harvest.</li>
	<li>Place the implant in 0.5 mL of 0.3% Tween-80 in TSB and sonicate for 15 min.</li>
	<li>Vortex the resulting implant suspension for 2 min and culture overnight for 12-16 h.</li>
	<li>Homogenize the soft tissue and spinous processes previously placed in 1 ml PBS surrounding the implant using homogenizer.</li>
	<li>Vortex the resulting soft tissue suspension for 5 min and culture overnight for 12-16 h.</li>
	<li>After overnight culture, count CFU from the implant and surrounding tissues, respectively. Express value as total CFU/g harvested for soft tissue and CFU/mL for sonicated implant.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Implant related infections in the spine portend poor outcomes for patients1,2,3,4,5. Unlike many other areas in the body, infected hardware in the spine frequently cannot be removed due to the risk of instability and neurologic compromise. This unique challenge in the setting of biofilm bacteria resistant to systemic antibiotic therapy necessitate novel approaches to treatment12. Previous research in novel treatments for SII has been limited by expensive, inefficient animal models. To better study these infections and efficiently assess the efficacy of potential treatments, we developed a noninvasive longitudinal mouse model of SII using in vivo bioluminescence imaging.
Prior animal models of instrumented spine infection required ex vivo tissue analysis to evaluate results, requiring large cohorts and were unable to monitor infection over time13,14,26. In contrast, the mouse model of SII presented in this paper leverages BLI to reliably monitor bacterial burden over time16,17,18. This novel approach enables investigators to assess the response of bacteria and the host to antibiotics, coatings, or immune modulation.
Critical steps in the protocol include: appropriate preparation of Xen36 S. aureus and estimation of single inoculum 1 x 103 CFUs/2 &#956;L; precise surgical implantation, inoculation, and closure; in vivo bioluminescent imaging; and confirmation of bacterial burden using CFU counts.
Modifications to the model may include alternative bioluminescent bacterial strains, longer term time points, or the use of integration of other types of genetically engineered mice, such as those expressing green fluorescent protein in myeloid cells (Lys-EGFP) to contemporaneously measure neutrophil infiltration with in vivo fluorescence imaging20. Additional methodologies may be utilized to complement those described in the protocol to address processes such as vertebral osteolysis, disc degeneration, soft tissue infection, and implant biofilm infection. Techniques that provide quantitative outcomes in these processes may include but are not limited to: micro-computed tomography, magnetic resonance imaging, real time quantitative PCR, serology, histology, immunohistochemistry, and/or variable pressure scanning electron microscopy.
This model has several limitations. First, the model of spine surgery is a gross simplification compared to clinical practice. In contrast to extensive decompression and multilevel fusion surgeries typical of high-risk spine surgery patients, the model surgery involves minimal bone resection with a single stainless-steel implant. As clinical spine implants have multiple different materials, these may have different susceptibilities to bacterial infection and biofilm formation. In addition, as with all animal models, the host response to infection of mice is different than that of humans.
In the future, this model could be used to assess treatment of SII with other antibiotic delivery modalities including vancomycin powder, antibiotic loaded beads, or coated implants. In addition, this model may be used to study the mechanistic basis of the host response to SII.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated from:
Benjamin V. Kelley1 
Stephen D. Zoller1 
Danielle Greig1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles
to:
Benjamin V. Kelley1 
Christopher Hamad1 
Stephen D. Zoller1 
Danielle Greig1 
Zeinab Mamouei1 
Rene Chun1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Brandon Gettleman3 
Autreen Golzar2 
Adrian Lin2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles 
3University of South Carolina School of Medicine, University of South Carolina

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated.

Erratum: In vivo Mouse Model of Spinal Implant Infection

The purpose of this method is to describe a novel mouse model of spinal implant infection (SII). This model was designed to provide an inexpensive and accurate tool to flexibly assess the effect of host, pathogen, and/or implant variables in vivo. Testing potential therapeutics and treatment strategies for spinal implant infections in this model is aimed at guiding research development prior to application in larger animal models and clinical trials.
Implant related infection after spine surgery is a devastating complication and unfortunately occurs in approximately 3&#8211;8% of patients undergoing elective spine surgery1,2,3,4,5 and up to 65% of patients undergoing multilevel or revision surgery6. Treatment of spinal implant infections often requires multiple hospitalizations, multiple surgeries, and prolonged antibiotic therapy. SIIs portend poor patient outcomes including neurological compromise, disability, and an increased risk of mortality. Management of SII is extremely expensive, costing upwards of $900,000 per patient7.
Staphylococcus aureus is the most common virulent pathogen of SII8,9,10,11. Bacteria can seed the hardware directly during surgery, through the wound during the postoperative period, or later via hematogenous spread. In the presence of metal implants, S. aureus form biofilm that protects the bacteria from antibiotic therapy and immune cells. While removal of infected hardware may help effectively eradicate an infection, this is frequently not feasible in the spine without causing destabilization and risking neurologic compromise12.
In the absence of explanting infected hardware, novel approaches are needed to prevent, detect, and treat SII. Historically, there have been limited animal models of SII to efficiently assess the safety and efficacy of novel therapies. Previous animal models of SII require large numbers of animals and collection of data points requiring euthanasia including colony counting, histology, and culture13,14,15. Lacking longitudinal in vivo monitoring, these models only provide one data point per animal and are therefore expensive and inefficient.
Previous work studying a mouse model of knee arthroplasty infection established the value and accuracy of noninvasive in vivo optical imaging to longitudinally monitor infection burden16. The detection of bioluminescence allows bacterial burden to be quantified over a longitudinal time course in a single animal humanely, accurately, and efficiently. Moreover, prior studies have demonstrated a high correlation between in vivo bioluminescence and CFUs adherent to implants17. The capacity to track infection over time, has led to a more nuanced understanding of implant related infection. In addition, monitoring longitudinal infection in this way, has allowed the effectiveness of antibiotic therapy and novel antimicrobials to be accurately assessed16,17,18.
Leveraging these tools, we developed and validated a model of postoperative spinal implant infection. In the method presented, we utilize an inoculum of bioluminescent S. aureus Xen36 to establish an in vivo mouse model of SII to longitudinally monitor bacterial burden16,17,18. This novel model provides a valuable tool to efficiently test potential detection, prevention, and treatment strategies for SII prior to their application in larger animal models and clinical trials.

<table><tbody><tr><td>Analytical Balance ME104</td><td>Mettler Toledo</td><td>30029067</td><td>120 g capacity, 0.1 mg readability, backlit LCD, internal adjustment, metal base</td></tr><tr><td>BD Bacto Tryptic Soy Broth</td><td>Becton Dickinson (BD)</td><td>BD 211825</td><td>BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium)</td></tr><tr><td>Biomate 3S UV-VIS Spectrophotometer</td><td>Thermo Scientific</td><td>840-208300</td><td>Spectrophotometer; Thermo Scientific; BioMate 3S; Six-position cell holder; Spectral bandwidth: 1.8nm; Long-life xenon lamp; Store up to 40 test methods; 16L x 13W x 9 in. H; 19 lb.; 100/240V US line cord</td></tr><tr><td>Bioshield 720+ swinging bucket rotor</td><td>Thermo Scientific</td><td>75003183</td><td>Rotor, Swinging bucket; Thermo Scientific; BIOShield 720 high speed; Capacity: 4 x 180mL (0.72L); Angle: 90 deg. ; Max. speed/RCF: 6300rpm/7188 x g; Max. radius: 16.2cm</td></tr><tr><td>Branson Ultrasonics 2510R-MTH (Sonicator)</td><td>Branson Ultrasonics</td><td>CPX952217R</td><td>*similar model, our model is discontinued* Branson Ultrasonics MH Series Heated Ultrasonic Cleaning Bath, 120V, 0.75 gal</td></tr><tr><td>Bullet Blender Storm Homogenizer</td><td>Next Advance</td><td>BBY24M</td><td>The Bullet Blender Storm is the most powerful member of the Bullet Blender family. Homogenize up to 24 of your toughest samples (mouse femur, skin, cartilage, tumor, etc.) in just minutes. Air cooling&amp;trade; minimizes sample heat up. Uses 1.5ml screw-cap RINO&amp;reg; tubes or snap-cap Eppendorf&amp;reg; Safe-lock&amp;trade; tubes.</td></tr><tr><td>Germinator 500</td><td>Electron Microscopy Sciences</td><td>66118-10</td><td>The Germinator 500 is designed to decontaminate metal micro-dissecting instruments only. It is to be&lt;br/&gt; used exclusively for research purposes. The Germinator 500 should not be used as a substitute for&lt;br/&gt; traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine&lt;br/&gt; sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been&lt;br/&gt; designed and built to pass the Validation of Dry Sterilizer Spore Suspension Test: USP XXIII, Part 1211.</td></tr><tr><td>Heracell 150i CO2 Incubator</td><td>Thermo Scientific</td><td>51026282</td><td>Single 150L</td></tr><tr><td>IVIS Lumina X5 Imaging System</td><td>Perkin Elmer</td><td>CLS148590</td><td>The IVIS Lumina X5 high-throughput 2D optical imaging system combines high-sensitivity bioluminescence and fluorescence with high-resolution x-ray into a compact system that fits on your benchtop. With an expanded 5 mouse field of view for 2D optical imaging plus our unique line of accessories to accelerate setup and labeling, it has never been easier or faster to get robust data&amp;mdash;and answers&amp;mdash;on anatomical and molecular aspects of disease.</td></tr><tr><td>MAXQ 4450 Digtial Incubating Bench Shaker</td><td>Thermo Scientific</td><td>SHKE4450</td><td>Shaker, Incubated; Thermo Scientific; Digital; MaxQ 4450; Speed 15 to 500rpm +/-1rpm; 5 deg. C above ambient to 80 deg. C; 120V 50/60Hz</td></tr><tr><td>PBS, Phosphate Buffered Saline</td><td>Fisher Bioreagents</td><td>BP24384</td><td>PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4</td></tr><tr><td>Sorvall Legend Micro 21 Centrifuge, Ventilated</td><td>Thermo Scientific</td><td>75002436</td><td>24 x 1.5/2.0mL rotor with ClickSeal biocontainment lid</td></tr><tr><td>SORVALL LEGEND X1R 120V Centrifuge</td><td>Thermo Scientific</td><td>75004261</td><td>Centrifuge, Benchtop; Thermo Scientific; Sorvall Legend X1R (Refrigerated), 1L capacity; Max. Speed/RCF 15,200rpm/25,830 x g; CFC-free cooling -10C to +40C; 120V 60Hz</td></tr><tr><td>Staphylococcus aureus - Xen36</td><td>Perkin Elmer</td><td>119243</td><td>Staphylococcus aureus - Xen36 bioluminescent pathogenic bacteria for in vivo and in vitro drug discovery. This product was derived from a parental strain from the American Type Culture Collection, used under license. Staph. aureus-Xen36 possesses a stable copy of the Photorhabdus luminescens lux operon on the native plasmid.</td></tr><tr><td>TUTTNAUER AUTOCLAVE 2540E 120V</td><td>Heidolph Tuttnauer</td><td>23210401</td><td>Sterilizer, Benchtop; Heidolph; Tuttnauer; Model 2540E; Self-contained design with refillable reservoir controls water purity for sterilization; 120V 50/60Hz; 1400w. With electronic controls</td></tr><tr><td>Tween 80</td><td>Fisher Bioreagents</td><td>BP338-500</td><td>Tween 80, Fisher BioReagents, Non-ionic detergent for selective protein extraction</td></tr><tr><td>Vortex mixer VX-200</td><td>Labnet Internation</td><td>S0200</td><td>120V touch or continuous mixer, 230V: 0 - 2,850 rpm,120V: 0 - 3,400 rpm</td></tr><tr><td>0.9% Sodium Chloride</td><td>Pfizer Injectables/Hospira</td><td>00409-4888-10</td><td>0.9% Sodium Chloride Injection, USP</td></tr></tbody></table>

in vivo mouse model of spinal implant infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The procedure presented here was used to assess the efficacy of antibiotic regimens in an in vivo mouse model of SII. Specifically, the efficacy of combination vancomycin and rifampin antibiotic therapy was compared to vancomycin monotherapy and untreated infected controls.
Prior to surgery, mice were randomized to either combination therapy, monotherapy, or infected control. A statistical power analysis was performed to calculate sample size. Anticipated means of mean maximum flux 1 x 105 &#177; 3.2 x 104 and 1.4 x 105 were used to determine sample size, which were calculated as N=10 in each group. Mice underwent surgical implantation, inoculation with S. aureus Xen36, and were measured for in vivo S. aureus bioluminescence on POD 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. On POD 35, mice were sacrificed and CFUs for implant-adherent and surrounding tissue bacteria were quantified.
Mice in the monotherapy group received a therapeutic dose of vancomycin (110 mg/kg twice daily) delivered subcutaneously. This dose was selected to approximate the area under the curve for typical human exposure for vancomycin21,22,23. Mice in the combination therapy group received a therapeutic subcutaneous dose of vancomycin (110 mg/kg twice daily) and rifampin (25 mg/kg daily)24. Mice in the infected control group received sham injections of sterile saline. Treatment for all groups was performed from postoperative days 7 to 14.
Effect of antibiotic therapy on BLI 
Infected control mice had BLI signals peaking on POD 10 that remained above 1.0 x 105 photons/s/cm2/sr until sacrifice, successfully modeling a chronic SII (Figure 2). Mice treated with vancomycin monotherapy had significantly lower BLI signal compared to infected control, with a 2-fold reduction from POD 10&#8211;21 (p&#60;0.03). After POD 21, there was no significant difference in BLI between monotherapy and infected control groups. Mice treated with vancomycin-rifampin combination therapy had an even lower BLI signal, which was 20-fold lower than infected control on POD 10. A significant reduction persisted until POD 28 (p&#60;0.01). After POD 28, there was no significant difference in BLI between groups. There was no significant difference in BLI between any of the three groups at final imaging on POD35.
CFUs from implants and surrounding tissue 
Mice were sacrificed on POD 35. Implants and surrounding tissue were harvested and processed for CFU counting (Figure 3). No significant difference was observed in CFUs between infected control, monotherapy, or combination therapy groups.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60560/60560fig1v2.jpg" /> 
Figure 1: Wound breakdown in high dose S. aureus Xen36 inoculum.&#160;Images of the dorsal skin of mice inoculated with S. aureus Xen36 during a spine implant infection. (A) Mouse inoculated with 1 x 103 CFUs, and intact dorsal skin. (B) Mouse inoculated with 1 x 104 CFUs, and evidence of considerable wound breakdown. Figure adapted and reprinted with permission from Dworsky et al.25 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60560/60560fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60560/60560fig2v2.jpg" /> 
Figure 2: Measurement of bacterial burden using in vivo bioluminescence.&#160;1 x 103 CFU of S. aureus possessing the bioluminescent construct in a stable plasmid (Xen36) were inoculated into the L4 spinous process of mice (n = 10 mice per group) in the presence of a stainless-steel implant. (A) Bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux [photons/s/cm2/sr] &#177; sem [logarithmic scale]), with a flow diagram of the experimental protocol below. On POD 7, antibiotic administration began with vancomycin, a combination of vancomycin and rifampin or a sterile saline control. Antibiotic administration was stopped on POD 14. On POD 35, mice were sacrificed and CFUs from the implant and surrounding tissue were measured. (B) Representative in vivo S. aureus bioluminescence on a color scale overlaid on top of a grayscale image of mice. Figure adapted and reprinted with permission from Hu et al.25 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60560/60560fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60560/60560fig03.jpg" /> 
Figure 3: Confirmation of bacterial burden using CFU counts.&#160;At POD 35, mice were sacrificed, pins were sonicated, tissue was homogenized, and bacteria were cultured and counted. Figure adapted and reprinted with permission from Hu et al.25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60560/60560fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

In Vivo Mouse Model of Spinal Implant Infection

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

in-vivo-mouse-model-of-spinal-implant-infection

Nanyang Technological University

Research

JoVE Journal

Medicine

2.3K Views.  University of California Los Angeles. Het protocol beschrijft een nieuw in vivo muismodel van spinale implantaatinfectie waarbij een roestvrijstalen k-draadimplantaat is geïnfecteerd met bioluminescente Staphylococcus aureus Xen36. De bacteriële belasting wordt longitudinaal gemonitord met bioluminescente beeldvorming en bevestigd met kolonievormende eenheidstellingen na euthanasie.

Video: In Vivo Mouse Model of Spinal Implant Infection

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

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Cancer Research

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

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Neuroscience

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

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Developing Efficient and Precise Mouse Model for Spinal Cord Glioma

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Immunology and Infection

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is characterized by inflammation in the tissues around dental implants with progressive loss of supporting bone. To optimize dental implants' longevity in terms of health and functionality, it is crucial to understand the peri-implantitis pathophysiology. In this regard, using mouse models in research has proven clear benefits in recreating clinical circumstances. This study aimed to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease, given the observed bone and tissue changes. The experimental peri-implantitis induction comprehends the following steps: teeth extraction, implant placement, and ligature-inducted PI. A sample of eighteen 3-week-old C57BL/6J male mice was divided into two groups, ligature (N=9) and control non-ligature (N=9). The evaluation of clinical, radiographical, and histological factors was performed. The ligature group showed significantly higher bone loss, increased soft tissue edema, and apical epithelial migration than the non-ligature group. It was concluded that this pre-clinical model can successfully induce peri-implantitis in mice.

This is a report on an experimental model of ligature-induced peri-implantitis in mice. We describe all surgical steps, from pre- and post-operative management of the animals, extractions, implant placement, and ligature-induced peri-implantitis.

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A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Biology

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

Source: Anderson, L. S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to generate a murine model of skin wound infection. Upon infecting mice &#8212; engineered to express a fluorescent protein in neutrophils &#8212; with a genetically modified bioluminescent Staphylococcus aureus &#8212; a pathogenic bacteria. The bacterial burden at the infection site and the neutrophil recruitment dynamics can be studied using whole animal imaging.

In Vivo Mouse Model of Spinal Implant Infection

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