The authors would like to thank Janelle Ryals, Rachel Supple, and Frank Wang for technical assistance. This work was supported by NIH grants R03 DK080182 (JAC), R01 DK099611 (JAC), R01 DK103872 (JAC), Center of Biomedical Research Excellence (COBRE) grant P20 GM104936 (JAC), start-up funds and core support from the Kansas Institutional Development Award (IDeA) P20 GM103418, core support from the Kansas IDDRC P30 HD002528, and The Madison and Lila Self Fellowship Program (ANP).

All experiments described in this protocol conform to NIH guidelines in accordance with the guidelines specified by the University of Kansas Medical Center Institutional Animal Care and Use Committee.
1. Neonatal Maternal Separation (NMS)
<ol>
	<li>Monitor pregnant dams daily for litter births. 
	Note: The day the litter is born is considered P0.</li>
	<li>On P1, remove the dam from the home cage and place into a clean secondary container.</li>
	<li>Rub a handful of bedding between clean-gloved hands to maintain scent of the home cage.</li>
	<li>Add a depth of 1 - 2 cm of the home cage bedding into a clean, appropriately labeled 2 L glass beaker. Add approximately half of any additional enrichment bedding material that is present in the home cage, e.g., nestlet, crinkle paper, to the glass beaker.</li>
	<li>Gently place all of the pups from a single litter, individually, in the same beaker.</li>
	<li>Immediately place the beaker in an incubator held at 33 oC and 50% humidity for 180 min.</li>
	<li>Remove the dam from the secondary container and return her to the home cage. Return the home cage to its appropriate housing location within the vivarium.</li>
	<li>Repeat this procedure for each litter undergoing NMS, using clean gloves and secondary containers for each litter/dam.</li>
	<li>At the end of the 180 min separation period, return the litters to their home cages in the same order as they were removed.</li>
	<li>Retrieve the home cage of the first litter that was separated earlier in the day. Remove the dam from the home cage and place into a clean secondary container.</li>
	<li>Remove the first beaker from the incubator and rub a handful of bedding between clean gloved hands to maintain scent of the home cage while handling the pups.</li>
	<li>Gently move the pups, individually, from the beaker to the home cage.</li>
	<li>Return any enrichment and bedding remaining in the beaker to the home cage.</li>
	<li>Return the dam from the secondary container to the home cage and return it to its appropriate housing location within the vivarium.</li>
	<li>Rinse the beaker with water and return it to the incubator. Use the same beaker for each litter throughout the separation procedure.</li>
	<li>Repeat this procedure for each litter undergoing NMS in the same order as they were placed in the incubator.</li>
	<li>Repeat this entire procedure for each litter undergoing NMS every day through P21.</li>
	<li>Wean NMS and na&#239;ve pups on P22 by placing littermates of the same sex together in clean cages complete with food and water. Na&#239;ve pups should be born and housed under the same conditions as NMS mice, but undergo no additional handling outside of normal animal husbandry tasks.</li>
</ol>
2. Perigenital Mechanical Sensitivity
<ol>
	<li>von Frey Apparatus Setup
	<ol>
		<li>Bring the mice to the testing room and allow them to acclimate for 30 min while remaining in their home cages.</li>
		<li>Prepare the testing area by placing an absorbent underpad beneath an elevated wire mesh-top table (79 cm x 28 cm) that provides enough space to allow the investigator to approach from the underside without startling the mice, approximately 55 cm in height.</li>
		<li>Place up to 12 mice, individually, under clear, perforated plastic chambers (11 cm x 5 cm x 3.5 cm) on top of the wire mesh table. Place a heavy object on top of the chambers to prevent mice from escaping.</li>
		<li>Acclimate the mice for an additional 30 min on the screen prior to withdrawal threshold assessment.</li>
	</ol>
	</li>
	<li>Perigenital Mechanical Sensitivity Assessment
	<ol>
		<li>Perform the up-down method using a standard set of graded von Frey monofilaments43. Use the following monofilaments: 1.65 g, 2.36 g, 2.83 g, 3.22 g, 3.61 g, 4.08 g, 4.31 g, and 4.74 g.
		<ol>
			<li>Hold the base of the 3.22 g monofilament and expose the retracted monofilament. 
			Note: Some models may not have a retracted monofilament.</li>
			<li>Situate the probe so that the monofilament is vertically oriented and when the mouse is alert, immobile, and its body weight is distributed evenly between the hind paws apply it to either the left or right side of the scrotum, avoiding the midline, until a slight bend is observed in the filament.</li>
			<li>Hold the monofilament in place for 10 sec or until the animal shows a positive response. 
			Note: A positive response is considered to be a brisk jerk or jump in response to monofilament application or licking or biting behavior directed towards the monofilament. If the mouse moves during the 10 sec monofilament application without displaying these behaviors, the monofilament must be retested following a minimum 1 min-long rest period. If a mouse neither moves nor exhibits any of above listed behaviors during the 10 sec monofilament application, it is considered a negative response.</li>
			<li>Record the response, either negative or positive, in a lab notebook or laptop and repeat this procedure on all of the remaining mice using the 3.22 g monofilament.</li>
			<li>Test all of the mice again using the next appropriate monofilament. Note: If the mouse exhibited a negative response to the 3.22 g monofilament, apply the 3.61 g monofilament in the same manner and record the response. If the mouse exhibited a positive response to the 3.22 g monofilament, apply the 2.83 g monofilament and record the response.</li>
		</ol>
		</li>
		<li>Continue testing each mouse using the next larger or smaller monofilament, as appropriate, for an additional 4 applications after the first positive response is observed.</li>
		<li>Return mice to home cages.</li>
		<li>Use the value of the final von Frey monofilament applied in the trial series in log units to calculate a 50% g withdrawal threshold as described in Chaplan et al.43.</li>
	</ol>
	</li>
</ol>
3. Acidified Toluidine Blue Mast Cell Staining
<ol>
	<li>Tissue Processing
	<ol>
		<li>Dissect prostate tissue44 from mice that have been intracardially perfused with ice-cold 4% paraformaldehyde45. Postfix the tissue in 4% paraformaldehyde for 1 hr at RT and then cryoprotect in 30% sucrose in 1x&#160;PBS at 4 &#176;C O/N.</li>
		<li>Prepare a small (30 ml) bath of heptane and place it on dry ice.</li>
		<li>Rinse the prostate tissue in 1x PBS and dry using a light duty wipe.</li>
		<li>Insert the prostate tissue into the cold heptane until it is frozen.</li>
		<li>Immediately place the frozen prostate tissue in a cryomold containing mounting media and place on dry ice until frozen.</li>
		<li>Store cryomolds at -20 &#176;C.</li>
		<li>Transversely cut the prostate tissue into 7 &#181;m-thick cryosections using a cryostat.</li>
		<li>Place 8 - 12 non-serial cryosections that span the length of the tissue, onto positively-charged microscope slides.</li>
		<li>Store finished slides at -20 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Preparing Acidified Toluidine Blue
	<ol>
		<li>Add 1 g of Toluidine Blue O to 100 ml 70% ethanol and vortex until in solution to prepare a 1% stock solution. The stock solution can be stored at RT for up to three weeks.</li>
		<li>Add 1 g NaCl to 100 ml water in a beaker placed on top of a stir plate.</li>
		<li>Adjust the pH of the NaCl solution using 12 M HCl until a pH range of 0.5 - 1.0 is achieved.</li>
		<li>Prepare the 0.25% Toluidine Blue working solution by combining 8 ml of the Toluidine Blue stock solution and 32 ml of the 1% NaCl solution. Pipette up and down to ensure total incorporation of the Toluidine Blue stock solution and mix using a vortex. The working solution must be made fresh and discarded after using.</li>
	</ol>
	</li>
	<li>Staining Prostate Cryosections
	<ol>
		<li>Remove the slides to be processed from the freezer and allow to equilibrate to RT for approximately 30 min.</li>
		<li>Wash the tissue sections by individually dipping the slides for approximately 1 sec into a 50 ml conical tube containing a sufficient volume of 1x&#160;PBS to ensure that the slide-mounted tissue sections will be completely submerged. Allow slides to air dry on a paper towel.</li>
		<li>Place slides in a glass staining jar containing enough 0.25% working Toluidine Blue solution to ensure that the slide-mounted tissue sections will be completely submerged and incubate for 10 - 15 min while on a platform rocker set at 15 rpm.</li>
		<li>Dip slides in 1x PBS for approximately 1 sec to wash out excess Toluidine Blue.</li>
		<li>Place slides in a slide box or rack in such a way that the slides are on their sides to allow for drainage.</li>
		<li>Dry the slides in an oven at 37 &#176;C for a minimum of 2 hrs or O/N at RT.</li>
		<li>Dehydrate slides quickly using alcohol, allowing the slides to completely dry between alcohol exposures. Dry slides by draining on a paper towel and wiping backs and edges with a light duty wipe.
		<ol>
			<li>Immerse the slides in 95% EtOH for approximately 1 sec and allow to completely dry. Repeat this procedure 1 - 10 times until tissue dries more blue than purple.</li>
			<li>Immerse the slides in 100% EtOH for approximately 1 sec and allow to completely dry. Repeat this procedure 1 - 10 times until tissue is dark to medium blue, but not purple.</li>
		</ol>
		</li>
		<li>Fix the stain by incubating the slides in 100% xylene for 3 min. Allow to dry.</li>
		<li>Coverslip the slides with glycerol, PBS, or xylene-based permanent mounting media. Drain excess media and store at RT.</li>
	</ol>
	</li>
	<li>Quantifying Mast Cells
	<ol>
		<li>Visualize the toluidine blue-stained tissue sections at 20X magnification using light microscopy (Figure 2).</li>
		<li>Count the total number of mast cells present within the visualized area and differentiate between non-degranulated and degranulated mast cells. 40X magnification may be necessary to confirm granulation status in some mast cells. 
		Note: Non-degranulated mast cells exhibit dense metachromasia with no or faint nuclear outline and/or no granular extrusion around the cell. Degranulated mast cells exhibit less intense metachromasia and have an obvious clear outline of the nucleus and/or free granules within the cytoplasm and outside of the cell border.</li>
		<li>Continue to assess the total number of non-degranulated and degranulated mast cells in the entirety of the current tissue section. Repeat this procedure on at least 7 additional separate sections, spanning the length each tissue.</li>
		<li>Calculate the percentage of degranulated (DG) to total mast cells for each tissue/mouse according to the following equation: (DG mast cells / Total mast cells) x 100.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol provides methodology for using neonatal stress, in the form of NMS, to induce symptomology indicative of CP/CPPS in adult male mice. As adults, the NMS mice display significant perigenital mechanical allodynia, as well as evidence of mast cell degranulation in prostate tissue. The use of NMS is a novel approach to developing a preclinical model of CP/CPPS in that it replicates the early life stress that is often reported by patients with chronic pelvic pain12-14, as well as incorporating a non-in...

Chronic pelvic pain is not in itself a disease, but rather a term associated with the ongoing spontaneous and/or evoked pain experienced by patients diagnosed with irritable bowel syndrome (IBS), interstitial cystitis/painful bladder syndrome (IC/PBS), vulvodynia, or chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). These syndromes are often comorbid and share many characteristics in that they have no associated pathology or identified underlying etiology, although dysfunction within the immune system, central nervous system, and peripheral nervous system has been shown to contribute towards the maintenance and progression of these disorders1-3. Patients with chronic pelvic pain are more likely to present with symptoms of additional, non-pelvic-related functional pain disorders and mood disorders, including anxiety, depression, and panic disorder4-6, which has been associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis7-10. Exposure to early life stress or trauma is a significant risk factor for developing HPA abnormalities and associated chronic pain syndromes10,11 and, as such, a significant subset of patients with functional pelvic pain disorders report having experienced adverse childhood events such as abuse or neglect12-14.
Rodent models of neonatal maternal separation (NMS), which involves removing the pups from their dam for a set period of time during the preweaning period, have been used for the past two decades to study the outcomes of early life stress. In general, NMS has been shown to increase HPA axis activation, and resultant anxiety-like behaviors, by directly affecting gene expression within the hypothalamus, as well as disrupting downstream regulation from limbic structures15-18. Disruption of proper HPA axis functioning has been shown to contribute towards increased colorectal19-22 and vaginal16 sensitivity displayed by rodent NMS models, but despite extensive characterization of the long-term effect of postnatal bladder inflammation23-25, the impact of early life stress has largely gone unstudied in the urogenital organs. Therefore, the following study will describe how to perform NMS in mice and later evaluate perigenital mechanical sensitivity and mast cell infiltration/activation in the prostate to validate the use of male NMS mice as a preclinical model for CP/CPPS.
Of all of the diagnosed chronic pelvic pain disorders, CP/CPPS is perhaps the least well-recognized and characterized syndrome, despite having a lifetime prevalence of approximately 14%&#160;26 and annual patient costs estimated at $4,400 (twice that of low back pain or rheumatoid arthritis27). Patients with CP/CPPS report pain in the perineum, rectum, prostate, penis, testicles, and/or abdomen28, experience a higher degree of psychological stress than control patients29, and commonly present with symptoms of or are diagnosed with comorbid chronic pelvic pain or mood disorders5,29-31. Recurrent infection, leaky epithelium, neurogenic inflammation, and autoimmunity have all been surmised as potential underlying causes of CP/CPPS2,32,33, as well as mast cell activation and degranulation34. Expressed prostatic secretions from men with CP/CPPS had increased mast cell tryptase and nerve growth factor (NGF) levels34, and a later study confirmed that tryptase and carboxypeptidase A (CPA3), a marker of mast cell activation, were also increased in the urine of CP/CPPS patients35. The potential role for mast cells in the onset and maintenance of CP/CPPS has been a major focus of animal research on this syndrome thus far. The most commonly employed rodent model used to study CP/CPPS is an experimental autoimmune prostatitis (EAP) model generated by subcutaneous injection of prostate antigen in Complete Freund&#8217;s adjuvant, which results in varied degrees of prostatic inflammation depending on species and strain used34,36-39. Mast cell infiltration and activation/degranulation has been shown to increase following induction of EAP34,35,40. Transgenic mice deficient in either mast cells34 or the tryptase receptor PAR235 do not develop prostatic tactic sensitivity following EAP, unlike wildtype EAP mice. While this preclinical model replicates many of the characteristics of human CP/CPPS, the induction protocol is not indicative of the human condition, which has a diverse etiology and often does not involve direct inflammation, infection, or injury of the prostate.
The influence of early life stress on the development of CP/CPPS in humans has largely gone uninvestigated; however, a study by Hu, et al.41,&#160;demonstrated that men who reported a history of childhood physical, emotional, and/or sexual abuse were significantly more likely to experience symptoms suggestive of CP/CPPS. Furthermore, they showed that both pain and urinary scores were increased in patients with a history of physical and emotional abuse. We have previously demonstrated that the same NMS paradigm in female C57BL/6 mice produces vaginal hypersensitivity and abnormal gene expression in both the vagina and bladder suggestive of dysfunctional HPA axis output16. This evidence combined with the high prevalence of CP/CPPS patients presenting with other comorbidities42, including IC/PBS and mood disorders, that have more clearly been shown to be linked with early life stress exposure12-14, provides the rationale for using an NMS model to investigate CP/CPPS in mice.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 female mice&#160;</td>
			<td>Charles River</td>
			<td>027</td>
			<td>Timed or untimed pregnant females should be monitored daily for in-house birth.</td>
		</tr>
		<tr>
			<td>2L glass beaker(s)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS10002L</td>
			<td>Each NMS litter will be held in the same beaker throughout the 21 day separation period.</td>
		</tr>
		<tr>
			<td>VWR Forced Air Incubator, Basic</td>
			<td>VWR International</td>
			<td>414005-124</td>
			<td>The incubator should be held at 33&#176;C and 50% humidity.</td>
		</tr>
		<tr>
			<td>Touch Test Sensory Evaluator, Kit of 20 (Semmes-Weinstein Von Frey Aesthesiometer for touch assessment)</td>
			<td>Stoelting</td>
			<td>58011</td>
			<td>The following monofilaments should be used for perigenital sensitivity assessment: 1.65 g, 2.36 g, 2.83 g, 3.22 g, 3.61 g, 4.08 g, 4.31 g, and 4.74 g.</td>
		</tr>
		<tr>
			<td>Animal Enclosure (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>435</td>
			<td>Be sure to place a heavy object on top of the individual, acrylic animal enclosures to prevent mice from escaping.&#160;</td>
		</tr>
		<tr>
			<td>Mesh Stand for mice and rats (12 mice 6 rats)</td>
			<td>IITC</td>
			<td>410</td>
			<td>Place stand on a stable table top for comfortable access.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>Dilute the 10x PBS stock to 1x PBS.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>A 4% paraformaldehyde solution is needed to intracardially perfuse mice and postfix tissue in preparation for mast cell staining.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td>Cryoprotect fixed tissue in 30% sucrose solution at 4&#176;C overnight.</td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>246654</td>
			<td>Chill heptane on dry ice to freeze tissue.</td>
		</tr>
		<tr>
			<td>Standard Cryomold&#160;</td>
			<td>VWR International</td>
			<td>4557</td>
			<td>To assist in freezing prostate tissue prior to cryostat sectioning</td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>12 x 125 mL</td>
		</tr>
		<tr>
			<td>VWR Superfrost Plus Micro Slide</td>
			<td>VWR International</td>
			<td>48311-703</td>
			<td>75 x 25 x 1 mm</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Sigma-Aldrich</td>
			<td>198161</td>
			<td>Certified by the Biological Stain Commission. Suitable for use as a metachromatic stain for mast cells.</td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Biology Grade, Fisher Bioreagents; 95% and 100% solutions will be needed to dehydrate stained cryosections before mast cell visualization.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Acidified NaCl solution should be made fresh before staining cryosections.</td>
		</tr>
		<tr>
			<td>GeneMate Sterile 50 mL Centrifuge Tubes&#160;</td>
			<td>BioExpress</td>
			<td>C-3394</td>
			<td>Disposable tubes used to mix toulidine blue elements or dip slides into 1xPBS.</td>
		</tr>
		<tr>
			<td>Coplin staining dish for 10 slides, with ground glass cover</td>
			<td>Fisher Scientific</td>
			<td>08-815</td>
			<td>Hold up to 10 standard 3 x 1 in. (75 x 25mm) slides back-to-back. Use separate dishes for Toluidine Blue working solution, 95% EtOH, 100% EtOH, and xylene.</td>
		</tr>
		<tr>
			<td>Xylenes (Histological)</td>
			<td>Fisher Scientific</td>
			<td>X3P</td>
			<td>Once dehydrated, tissue is cleared with xylene.</td>
		</tr>
		<tr>
			<td>Microscope Slide Boxes, 100-Place</td>
			<td>VWR International</td>
			<td>82003</td>
			<td>Boxes can be used for storage and transportation of slides.</td>
		</tr>
		<tr>
			<td>Fisherbrand Microscope Slide Box</td>
			<td>Fisher Scientific</td>
			<td>22-363-400</td>
			<td>These slide boxes are typically small enough to fit inside a cryostate.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Glycerol is a traditional mounting medium.</td>
		</tr>
		<tr>
			<td>Mounting Medium, Richard-Allan Scientific</td>
			<td>VWR International</td>
			<td>4111</td>
			<td>Mounting medium firmly bonds the coverslip to the slide.</td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Rectangular, No. 1</td>
			<td>VWR International</td>
			<td>48393-106</td>
			<td>A coverslip is placed over the dehydrated and cleared tissue to protect the sample.</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>The Advanced Automated Research Microscope Eclipse 90i, used by our lab, has been discontinued and replaced Eclipse Ni-E.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

assessment of perigenital sensitivity and prostatic mast cell activation in a mouse model of neonatal maternal separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

Mice that have undergone NMS showed behavioral evidence indicative of CP/CPPS. When tested with a graded series of von Frey monofilaments, 8 week-old NMS mice (n = 4) displayed perigenital mechanical allodynia when compared to na&#239;ve counterparts (n = 5, Figure 2). This is evidenced as a significant reduction (p &#60; 0.01; Student&#8217;s t-test) in the mechanical withdrawal threshold that elicited a positive behavioral response, recorded as a brisk jerk or jump in response to monofilament ...

Watch this Scientific Journal Video about Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation at JoVE.com

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

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9.2K Views.  University of Kansas Medical Center. We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm – neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Video: Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

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

Intraoperative Assessment of Resection Margins in Oral Cavity Cancer: This is the Way

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery.
The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed.
The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

The goal of this protocol is to provide a clear overview of specimen-driven intraoperative assessment of resection margins. It is encouraged to implement this protocol to improve patient care at other institutes.