This work was supported by a grant from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) 1R21AR079153 and a University of Connecticut Research Enhancement Program (REP) grant. The authors would like to acknowledge the assistance of Renata Rydzik from the University of Connecticut MicroCT Imaging Core facility.

Precise Growth Plate Injury Study Using Trilineage Fluorescent Mice

<ol>
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The authors have no conflicts of interest to disclose.

The innovative use of tricolor collagen reporter mice enables the creation of growth plate defects with a predetermined size and location, significantly enhancing the accuracy of murine experimental models for growth plate injuries. Given the small size of the 2-week-old mice, it is critical to use a small 0.5 mm bur to create the injury to avoid weakening the limb and causing a full-thickness fracture. The surgeon must also apply just enough pressure when creating the defect to avoid drilling too deeply into the bone for the same reason. The use of the perioprobe is critical to confirming a consistent injury depth.
As with any surgery, it is important to confirm an adequate depth of anesthesia, confirmed by an occasional toe pinch and sterility is maintained throughout. Another surgical point of importance is that blunt dissection with a carver has been described because it avoids damaging soft tissue and helps to ensure the mice are able to ambulate immediately after recovery from anesthesia to reach the mother mouse for nutrition and comfort. In our experience, the wounds closed with sutures have remained successfully closed and wound clips are not required. Surgery on mice at 2 weeks of age is recommended to best mimic the young child that experiences growth plate fractures. One downside of this protocol is that given the unpredictable nature of birthing, the use of this mouse model requires the availability of the surgeon at short notice.
Regarding the positioning of the bur to create the defect, the protocol describes creating the injury using a mCherry/Texas red filter set that illuminates the hypertrophic zone within the growth plate because of the brightness of the collagen X fluorescence. To ensure the injury is created within the tibial growth plate, it is beneficial to slightly move the soft tissue opening to the left and right to confirm that the proximal tibial growth plate is in view, and not the femur. Switching between filter set channels to illuminate the proliferative chondrocyte zone or the adjacent bone sections is useful for confirming accurate placement relative to the location of the proliferative zone and adjacent bone sections.
While the proliferative chondrocyte zone and the epiphyseal and metaphyseal bone can be distinguished under fluorescence microscopy in the live mice, the real value of the Type II and Type I collagen reporters are realized during the histological analysis of the growth plate. Given the aqueous nature of cryo-histological processes, traditional chromogenic dye precipitation protocols are unsuitable due to the potential misalignment of color with fluorescent imaging caused by dehydration steps. Although the aqueous protocol yields staining patterns similar to those in paraffin sections, rapid post staining imaging is essential to prevent dye diffusion from the tissue. Utilizing 30% glycerol in distilled water as the mounting medium can decelerate this diffusion, allowing for multiple chromogenic staining on the same section, including cartilage with Safranin O/Fast Green.
The endochondral ossification process is clearly visible with red chondrocytes lining the evolving bony bridge (Figure 6). Additional use of immunohistochemistry techniques, for which there are many murine antibodies available, could further enhance mechanistic studies conducted in these transgenic mice. Altogether, the combination of faxitron, microCT, and cryo-histological imaging techniques in this transgenic mouse model offers a comprehensive understanding of macroscopic and microscopic changes that occur in response to growth plate injuries, paving the way for future therapeutic interventions to mitigate such adverse outcomes. Further genetic manipulations of these transgenic mice could be done to allow lineage tracing studies to understand the origin of the cells that are temporally and spatially involved in healing. Experimentation on mice with additional modifications would allow the study of cartilage diseases such as osteochondroma - an overgrowth of cartilage and bone near the growth plate.
The consistency of our model is demonstrated by the reproducible formation of bony bridges in all mice without needing to discard any mice from the group due to articular cartilage injury. This is an improvement over previous models that approached the growth plate from a cortical window below the growth plate and angled a sharp tool or bur upwards towards the growth plate and occasionally would overshoot into the articular cartilage. An additional injury of the articular cartilage does not mimic the commonly occurring growth plate injuries in children. The more precise injury of this animal model reduces the number of mice required per experiment and that is another improvement. The use of transgenic mice allows the researcher to focus the injury on sub-sections of the growth plate, such as the hypertrophic/provisionally calcified area or the epiphysis/resting zone/proliferative zone area, without affecting the articular cartilage. However, a limitation of this model is the variability in bony bridge volume, which can differ by up to 30% among injured animals. Consequently, detecting a clinically significant effect on bony bridge formation still necessitates a large number of animals to achieve statistical relevance.
Benefits to a mouse model as described here as compared to rat or rabbit growth plate injury models previously published7,9,10,14, include a lower number of animals used, cost reduction, an efficient replicate size due to reproducible bony bar formation, a shorter study time frame, and more precise injury placement due to live imaging of the triple transgenic mice. While not discussed in detail, this mouse model can be used to test tissue-engineered implants or biomaterials delivering growth factors. A notable limitation of this murine method is that the size of an implant used to deliver therapeutic drugs or cells is limited to the defect volume of roughly a 0.5 mm diameter sphere. Only larger animal models can accommodate the volume of test material that would be used in human patients. The bur defect created in this protocol is not the same geometry as a thin fracture and thus differs from actual human injuries. Nonetheless, the benefits of this mouse model are many, and the lateral approach avoids damaging the articular cartilage that would occur when approaching blindly above or below the growth plate in line with the tibial long axis. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

Bone growth plates play a pivotal role in the longitudinal growth of long bones during childhood and adolescence1. Situated at the ends of long bones, the growth plate comprises multiple zones, with chondrocytes being the key cellular components responsible for producing and maintaining this dynamic growth area. Endochondral ossification of the growth plate occurs to lengthen and expand the bones through a sequential progression of chondrocyte proliferation, hypertrophy, apoptosis, invasion by blood vessels, recruitment of osteoprogenitor cells, and finally, bone formation2. Since the growth plate is relatively softer than bone, it is highly susceptible to fracture when bones are overloaded during sports or other activities. The Salter-Harris classification outlines five distinct types of growth plate injuries3. The Type II fracture through the hypertrophic zone of the growth plate and the adjacent lower bone tissue is the most prevalent4. A bony bridge often forms in response to injuries of the hypertrophic zone or the adjacent bone and leads to premature fusion of the adjacent long bone sections5. Bony bridges impede the normal expansion of the growth plate. Currently, no preventative treatments are available for the bony bridge formation, and some are left untreated depending on the patient&#39;s age and the bony bridge size and location6. When limb malformation is severe, surgical options include removal followed by implanting interpositional materials like fat or silicone rubber or corrective osteotomy and bone lengthening procedures; yet a bony bridge may still reform6. More research is needed to prevent bony bridge formation and to improve the outcomes of children with bone growth plate injuries.
Several animal models have been established to explore the underlying mechanisms and develop new strategies to prevent bony bridge impairment of growth plates post injury7,8,9,10,11,12. These animal models frequently focus on the proximal tibial growth plate and distal femur growth plate as the primary injury site, given this is typically where human injuries occur. The animal bone defects are created either by a lateral approach similar to an actual fracture pathway or an approach from above or below the growth plate leading to a central drill hole in the growth plate. In a previously reported rat model, a growth plate defect is created by inserting a dental bur through a cortical window in the tibial midshaft and drilling upwards through the marrow towards the knee joint to centrally injure the growth plate7,13. Alternatively, a recent mouse model uses a lateral approach with a small-bore needle to create a planar needle track through the growth plate8. In a widely used rat model, the defect is created in the growth plate of the distal femur by drilling through the articular cartilage between the condyles9,14. In larger animals like rabbits and sheep, growth plate defects have been both laterally induced directly in the proximal tibia and the distal femur by drilling or cutting into the growth plate or by approaching from below and creating a central defect leaving the edges of the growth plate unaltered10,11,12,15.
Murine models for growth plate injuries are advantageous for mechanistic studies that can be accomplished with genetically modified mice, such as stem cell lineage tracing studies8. However, a significant challenge in murine or rat animal models is achieving consistent and precise injury to a particular sub-region of the growth plate. Injury of particular zones of the growth plate and adjacent bone is required to mimic one of the clinically relevant fracture paths described by the Salter-Harris classifications. The challenges to date in rodent models are primarily due to the lack of a visual means of identifying the substrata of the growth plate during the surgical creation of the injury. This protocol describes a refined technique for creating growth plate defects in targeted substrata of the murine growth plate by utilizing triple transgenic mice that express collagen I, II, and X fluorescent reporters16,17,18. The different colored fluorescence of these collagens in each of the primary zones of the growth plate allows visual discrimination of the various sections of the growth plate under a fluorescence stereo microscope during the surgical creation of the growth plate injury. The use of these transgenic mice allows for unprecedented injury accuracy in a young mouse at a comparable developmental stage to the children that are injured.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>2-methyl-butane</td>
			<td>Sigma Aldrich</td>
			<td>M32631</td>
			<td></td>
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			<td>Alcohol antiseptic pads</td>
			<td>Acme United Corporation</td>
			<td>H305-200</td>
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			<td>Axio Scan.Z1</td>
			<td>Carl Zeiss AG</td>
			<td>Axio Scan.Z1</td>
			<td></td>
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			<td>AxioVision software</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
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			<td>Betadine solution (10% povidone-iodine)</td>
			<td>Avrio Health L.P.</td>
			<td>67618-150-01</td>
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			<td>Calcein</td>
			<td>Sigma Aldrich</td>
			<td>C0875</td>
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			<td>Calcein Blue</td>
			<td>Sigma Aldrich</td>
			<td>M1255</td>
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			<td>CFP filter set</td>
			<td>Chroma Technology Corp.</td>
			<td>49001</td>
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			<td>Cryomatrix</td>
			<td>Thermo Scientific</td>
			<td>6769006</td>
			<td></td>
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			<td>Cryomolds</td>
			<td>Fisher Scientific</td>
			<td>Fisherbrand #22-363-554</td>
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			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>3050s</td>
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			<td>Cryostat blades</td>
			<td>Thermo Scientific</td>
			<td>3051835</td>
			<td></td>
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			<td>Cryotape</td>
			<td>Section Lab</td>
			<td>Cryofilm 2C</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved fine scissor&#160;</td>
			<td>Fine Science Tools</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved mosquito hemostatic forceps</td>
			<td>HuFriedyGroup</td>
			<td>H3</td>
			<td></td>
		</tr>
		<tr>
			<td>cy5 filter set</td>
			<td>Chroma Technology Corp.</td>
			<td>49009</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI filter set</td>
			<td>Chroma Technology Corp.</td>
			<td>49000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental bur (0.5 mm diameter)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cleoid discoid carver</td>
			<td>ACE Surgical Supply Inc.</td>
			<td>6200097A-EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry glass bead sterilizer (Inotech Steri 350)</td>
			<td>Inotech Bioscience, LLC</td>
			<td>IS-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear punch</td>
			<td>Fine Science Tools</td>
			<td>24212-01</td>
			<td></td>
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		<tr>
			<td>Electric heating pad</td>
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			<td>Electronic foot control</td>
			<td>Nouvag AG</td>
			<td>1866nou</td>
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			<td>Electronic motors 31 ESS</td>
			<td>Nouvag AG</td>
			<td>2063nou</td>
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			<td>Environmental surface barrier (3 x 12 inch tube sox)</td>
			<td>Patterson Companies, Inc.</td>
			<td>BB-0312H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethiqa XR (buprenorphine extended-release injectable suspension) 1.3 mg/mL</td>
			<td>Fidelis Animal Health</td>
			<td>86084-100-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Faxitron x-ray cabinet</td>
			<td>Kubtech Scientific</td>
			<td>Parameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Stereomicroscope</td>
			<td>Carl Zeiss AG</td>
			<td>Lumar V12</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP filter set</td>
			<td>Chroma Technology Corp.</td>
			<td>49020</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>ARK2183</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Thermo Scientific</td>
			<td>3051</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
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		<tr>
			<td>Handpiece (contra angle 32:1 push button)</td>
			<td>Nouvag AG</td>
			<td>5201</td>
			<td></td>
		</tr>
		<tr>
			<td>Implantology/oral surgery system control unit (Straumann)</td>
			<td>Nouvag AG</td>
			<td>SEM&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant sealing sterilization pouch with dual internal/external process indicators (3 1/2 x 5 1/4&#160; inch)</td>
			<td>Fisher Scientific</td>
			<td>01-812-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant sealing sterilization pouch with dual internal/external process indicators (5 4/1 x 10 inch)</td>
			<td>Fisher Scientific</td>
			<td>01-812-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (29 G)</td>
			<td>Exel International</td>
			<td>26028</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Dechra Pharmaceuticals plc</td>
			<td>17033-091-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane anesthetic system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry filter set</td>
			<td>Chroma Technology Corp.</td>
			<td>39010</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissor</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle (20 G)</td>
			<td>Becton, Dickinson and Company</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>HuFriedyGroup</td>
			<td>NHCW</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral buffered formalin (10%)</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-sterile applicator swabs</td>
			<td>Allegro Industries</td>
			<td>205</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven gauze (3 x 3 inch)</td>
			<td>Fisher Scientific</td>
			<td>22028560</td>
			<td></td>
		</tr>
		<tr>
			<td>Norland Optical Adhesive, 61</td>
			<td>Norland Optical</td>
			<td>Norland Optical Adhesive, 61</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment (Optixcare eye lube)</td>
			<td>CLC Medica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodontal probe</td>
			<td>HuFriedyGroup</td>
			<td>PQW</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) pH 7.4 (1x)</td>
			<td>Gibco, by Life Technologies</td>
			<td>10-010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic microscope slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>71890-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional clipper/trimmer (Wahl Classic Peanut)</td>
			<td>Wahl Clipper Corporation</td>
			<td>8685</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-46</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanco Medical software</td>
			<td>SCANCO Medical</td>
			<td>Scanco &#956;CT 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Sigma Aldrich</td>
			<td>S2252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tartrate dibasic dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>T6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen disc</td>
			<td>Leica Biosystems</td>
			<td>14037008587</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel #15 surgical blade&#160;</td>
			<td>Aspen Surgical Products, Inc.</td>
			<td>371615</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>Cardinal Health, Inc.</td>
			<td>2D72PT65X</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile towel drape (18 x 26 inch)</td>
			<td>IMCO</td>
			<td>4410-IMC</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>Becton, Dickinson and Company</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Undyed braided coated vicryl suture (5-0)</td>
			<td>Ethicon Inc.</td>
			<td>J490G</td>
			<td></td>
		</tr>
		<tr>
			<td>UV black light</td>
			<td>General Electric</td>
			<td>F15T8-BLB</td>
			<td></td>
		</tr>
	</tbody>
</table>

tricolor transgenic murine model for studying growth plate injury

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones are overloaded.&#160;Better treatments for severely fractured growth plates are needed because the response to injury is a bony bridge that prematurely fuses the growth plate, leading to stunted and/or crooked limbs. Murine models of growth plate injury are advantageous for mechanistic studies, but&#160;are challenging because it is difficult to visualize and precisely injure the small growth plates in young mice.&#160;We describe here an improved growth plate injury model using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X.
These mice show native fluorescence associated with the three primary substrata of the growth plate. A growth plate injury similar to a Salter-Harris Type II injury is created reproducibly with a bur using the hypertrophic section of the growth plate as a reference during live imaging under fluorescence stereo microscopy guidance. Frozen histology analysis of the native fluorescence simplifies assessing the cellular response to injury. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

This protocol utilizes trilineage fluorescent reporter mice to induce a lateral growth plate defect in the proximal tibia with precision by leveraging the inherent red fluorescence emitted by type X collagen for surgical guidance. The view the surgeon has while looking through the stereomicroscope eyepiece with the mCherry filter set is shown in Figure 2D. The native Type X fluorescence allows the surgeon to place the bur in the hypertrophic zone and create an injury that mimics a common type of growth plate injury leading to a bony bridge (Figure 2F). The fluorescence under the red channel is the brightest and, therefore, recommended for use during bur placement. Alternatively, defect creation could be guided by using other colors of the native fluorescence of the triple transgenic mice if the goal of the experiment is to study injuries to other zones of the growth plate than the hypertrophic zone and adjacent calcified region.
The creation of a Salter-Harris Type II-like defect in the hypertrophic zone of the growth plate and the adjacent lower bone tissue, using a 0.5 mm diameter dental bur, was validated through microCT and cryo-histology imaging of the injured (time 0) proximal tibias compared to the uninjured lateral controls in N = 3 mice (Figure 4). The defects were difficult to see in the 3D microCT images but were detectable in the 2-D cross sections (Figure 3A,B,E,F). Figure 3G displays the distribution of type I collagen-producing bone cells (green fluorescence), type II collagen-producing proliferative chondrocytes (cyan fluorescence), and type X collagen-producing hypertrophic chondrocytes. In the image of the injured mouse (Figure 4G), there is a disruption of the hypertrophic zone, the provisionally calcified layer, and some of the newest formed bone relative to the control with the proliferative zone only slightly disturbed. Safranin O/Fast Green staining (Figure 4H) best illustrates the location of the defect within the injured growth plate since all cells are clearly visible.
X-ray analysis provides some insight into live mice as to the impact of this type of growth plate injury on tibia length and bony bridge formation over time (Figure 3). Comparative imaging between uninjured (Figure 3A) and injured (Figure 3B) tibiae, taken before surgery and 3 weeks post surgery, reveals a large amount of limb growth, thinning of the growth plates, and a distinct opaque region that has developed in the injured growth plate area at 3 weeks. This opacity within the growth plate is not present in the uninjured counterpart nor the mice before surgery. Faxitron is thus one way of observing pathological changes induced by the injury in live mice, such as the formation of a bony bridge and changes in limb length.
MicroCT imaging of dissected bones offers a detailed visualization of bony bridge formation within the injured growth plates three weeks after surgery (Figure 5). As seen in the images from six different injured mice shown in Figure 5, there is consistent bony bridge development in all mice. Utilizing Scanco Medical software, the bony bridge volume was calculated by reviewing each section of the proximal tibial growth plate, delineating the area of the bony bridge (Figure 5B) with the select tool, and then, integrating each section area throughout the entire growth plate volume to get the total volume24. The bony bridge volume calculated this way was 0.0761 mm3 &#177; 0.0246 (mean &#177; standard deviation, N = 6). The majority of the bony bridges form near the middle of the growth plate despite the lateral approach, which injures the outer edge as well as the center of the growth plate. This phenomenon can be attributed to the fact that mesenchymal stem cells (MSCs) from the bone marrow, rather than the perichondrium, are responsible for bony bridge formation25.
In these tricolor transgenic mice, cryo-histological analysis of the injured growth plate is enriched by the native collagen fluorescence (Figure 6). It reveals the complex interplay of bone cells and chondrocytes at the injury site. MicroCT images shown in Figure 6J,K were provided to the histology technician to guide the embedding and sectioning. The type I collagen-producing bone cells are seen in Figure 6L,O,P (green fluorescence), while type II collagen-producing proliferative chondrocytes are seen in Figure 6L,O,Q (cyan fluorescence). Type X collagen-producing hypertrophic chondrocytes are seen in Figure 6L,O,R (red fluorescence). This multicolor fluorescence approach enables a detailed examination of postsurgery chondrocyte differentiation within the bony bridge area against a backdrop of mineralized tissue. DAPI staining was used to confirm the distribution of all cell types within the growth plate area (Figure 6M). The Safranin O/Fast Green staining demonstrates the composite and structural organization of cartilage and bone within the injured growth plate (Figure 6N). Imaging these stained sections under a Cy5 filter set notably brightens the resting zone cells at the interface between the epiphyseal bone and cartilage.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66841/66841fig03.jpg" /> 
Figure 3: X-ray images of contralateral control and injured mouse tibiae. (A) X-ray images of the contralateral control tibia are taken just before injury when mice are 2 weeks old and at 3 weeks after surgery when the mice are 5 weeks old, demonstrating the extent of growth that occurs during this period. (B) Injured tibia from the same mouse at the same time points as in (A). The landmarks used for tibia length measurements are the apex of the proximal tibia head to the end of the tibia at the ankle joint (red double-headed arrows). The opaque bony bridge is visible in the injured proximal tibia growth plate at 5 weeks. Scale bars = 5.00 mm. <a href="https://www.jove.com/files/ftp_upload/66841/66841fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66841/66841fig04.jpg" /> 
Figure 4: MicroCT and histological images of time zero contralateral control and injured mouse proximal tibiae. (A,E) and (B,F) depict 3D and transverse 2D microCT views, with the defect indicated by red arrows in (E) and (F). (C,G) Composite merged cryo-histological images merging three innate fluorescence layers with a mineralized tissue layer. Green cells (Col3.6GFPtpz) are the type I collagen-producing bone cells, cyan blue colored cells (Col2A1GFPcyan) are type II collagen-producing proliferative chondrocytes, and red cells (Col10A1RFPchry) are type X collagen-producing hypertrophic chondrocytes. (D,H) Safranin O/Fast Green staining of the same region as (C) and (G). Scale bars = 1.0 mm. <a href="https://www.jove.com/files/ftp_upload/66841/66841fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66841/66841fig05.jpg" /> 
Figure 5: MicroCT images of bony bridges formed by this protocol. (A,C,E,G,I,K) Transverse cross-sections of the proximal tibial growth plate of six different mice at 3 weeks after bur defect creation. Bony bridge outlined by a red dotted line in (A). (B,D,F,H,J,L)&#160;3D reconstructions with a longitudinal plane cut away. Bony bridge outlined by a red dotted line in (B). Scale bars = 1.0 mm. <a href="https://www.jove.com/files/ftp_upload/66841/66841fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66841/66841fig06.jpg" /> 
Figure 6: MicroCT and histological images of contralateral control and injured mouse proximal tibia with bony bridge formation. (A,J) and (B,K) depict 3D and transverse 2D microCT views, with the bony bridge indicated by yellow arrows in (J) and (K). (C,L) Composite merged cryo-histological images merging three innate fluorescence layers with a mineralized tissue layer. Green cells (Col3.6GFPtpz) are the type I collagen-producing bone cells, cyan blue colored cells (Col2A1GFPcyan) are type II collagen-producing proliferative chondrocytes, and red cells (Col10A1RFPchry) are type X collagen-producing hypertrophic chondrocytes. The white box indicates the higher magnification shown in panels F and O. (D,M) The mineralized tissue and DAPI staining in the growth plate area of panels C and L. (E,N) Safranin O/Fast Green staining of the same region as (D) and (M) scanned with cy5 fluorescence. (F,O) A higher magnification of the growth plate area in the merged image of panels C and L. (G-I,P-R) Individual channels of the native fluorescence shown with a mineralized tissue backdrop. Scale bars = 1.0 mm (A-E) and (J-N), = 250 &#181;m in (F-I) and (O-R). <a href="https://www.jove.com/files/ftp_upload/66841/66841fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope.

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones ...

tricolor-transgenic-murine-model-for-studying-growth-plate-injury

Research

JoVE Journal

Medicine

476 Views.  University of Connecticut Health Center. This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope. 

Video: Tricolor Transgenic Murine Model for Studying Growth Plate Injury

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.