This research was partially supported by the Japan Agency for Medical Research and Development (AMED) under the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) project (Grant Number JP23dm0207001).

The present study was performed with the approval (W2023-2-041) of the Animal Experiments Committee of RIKEN (Saitama, Japan) and was conducted in accordance with the Institutional Guidelines for Experiments using Animals. In the present study, six female marmosets (2-6 years old) were used.
1. Criteria for commencement of treatment
<ol>
	<li>Start treatment when any one of the following criteria is observed: i) body weight &#60; 300 g or a decrease of &#62;20 g in the body weight per month; ii) hypoalbuminemia (serum albumin &#60; 3.8 g/dL); iii) anemia (hematocrit &#60; 35%); iv) chronic diarrhea without Clostridium difficile, enteropathogenic Escherichia coli, or intestinal protozoa; and v) an apparent decrease in muscle mass. 
	NOTE: Because of dehydration and subsequent hemoconcentration accompanied by WMS, the values of serum albumin and hematocrit may be artificially elevated in affected animals.</li>
</ol>
2. Oral administration
<ol>
	<li>Mix 0.1 mL of the iron formulation (see the Table of Materials) with a piece of sponge cake.</li>
	<li>Give the mash to the treated marmoset. 
	NOTE: Ensure that the treated marmoset has completely swallowed the mash.</li>
</ol>
3. Subcutaneous administration
<ol>
	<li>Catch the WMS-affected marmoset in the home cage by holding the tail gently.</li>
	<li>Hold the animal&#39;s upper body from the back with the other hand.</li>
	<li>Bring the animal to the treatment room.</li>
	<li>Let the animal cling to the assistant&#39;s chest or arm.</li>
	<li>Clean the area of the animal&#39;s back with an alcohol swab.</li>
	<li>Inject 0.1 mL of tranexamic acid (5% solution, see the Table of Materials) into the back subcutaneously using a 26 G needle 5x per week.</li>
	<li>Inject 5.0 mL of Ringer&#39;s lactate (see the Table of Materials) with 0.1 mL of a vitamin formulation (see the Table of Materials) subcutaneously using a 26 G needle 3x per week. 
	NOTE: Each injection should be done at each different spot within 1 min.</li>
</ol>
4. Intravenous administration of amino acid formulation
<ol>
	<li>Catch the WMS-affected marmoset in the home cage by holding the tail gently.</li>
	<li>Hold the animal&#39;s upper body from the back with the other hand.</li>
	<li>Bring the animal to the treatment room.</li>
	<li>Have the assistant restrain the animal&#39;s upper body by holding the base of both arms and restrain the lower body by holding the base of both legs with the other hand.Ask the assistant to place the thumb restraining the animal&#39;s lower body on the anterior side of the animal&#39;s knee to prevent it from bending.</li>
	<li>Clean the animal&#39;s calf with an alcohol swab.</li>
	<li>Inject 2.0 mL of the amino acid formulation (see the Table of Materials) intravenously via the saphenous vein using a 27 G butterfly needle three times per week. 
	NOTE: Injection must be done as slowly as possible up to 2 min.</li>
	<li>After injection, ask the assistant to press the area with absorbent cotton until bleeding stops.</li>
</ol>
5. Body weight measurement
<ol>
	<li>After treatment (intravenous and/or subcutaneous injection), weigh the animal with a carrying box once a week to monitor the body weight. 
	NOTE: Weigh the marmoset at around the same time because the body weight is affected by the presence of food.</li>
</ol>
6. Complete blood count (CBC) analysis and serum chemistry tests
<ol>
	<li>Catch the WMS-affected marmoset in the home cage by holding the tail gently.</li>
	<li>Hold the animal&#39;s upper body from the back with the other hand.</li>
	<li>Bring the animal to the treatment room.</li>
	<li>Have the assistant restrain the animal&#39;s upper body by holding the base of both arms and restrain the lower body by holding the base of either leg with the other hand.</li>
	<li>Ask the practitioner to hold the animal&#39;s leg that is not restrained.</li>
	<li>Clean the animal&#39;s inguinal region with an alcohol swab.</li>
	<li>Collect 0.4 mL of blood from the femoral vein using a 26 G needle monthly.</li>
	<li>Use 0.05 mL of the blood sample for CBC analysis, including hematocrit.</li>
	<li>Let the rest of the blood sample stand for 1 h at room temperature and centrifuge at 1,800 &#215; g for 20 min.</li>
	<li>Use the serum for chemistry tests, including albumin. 
	NOTE: The volume of the blood sample can be reduced by minimizing the number of parameters for the serum chemistry test. Adjust the volume of the blood sample according to the individual marmoset&#39;s condition.</li>
</ol>

An Efficient Modified Method for Treating Wasting Marmoset Syndrome

<ol><li>Baxter, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+albumin+and+body+weight+as+biomarkers+for+the+antemortem+identification+of+bone+and+gastrointestinal+disease+in+the+common+marmoset%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Serum albumin and body weight as biomarkers for the antemortem identification of bone and gastrointestinal disease in the common marmoset.</a> PLoS ONE. 8 (12), e82747(2013).</li>
<li>Brack, M., Rothe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+tubulointerstitial+nephritis+and+wasting+disease+in+marmosets+%28Callithrix+jacchus%29%5BTitle%5D)+AND+%22Vet+Pathol%22%5BJournal%5D)">Chronic tubulointerstitial nephritis and wasting disease in marmosets (Callithrix jacchus).</a> Vet Pathol. 18, Suppl 6 45-54 (1981).</li>
<li>Chalifoux, L. V., Bronson, R. T., Escajadillo, A., McKenna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+analysis+of+the+association+of+gastroenteric+lesions+with+chronic+wasting+syndrome+of+marmosets%5BTitle%5D)+AND+%22Vet+Pathol+Suppl%22%5BJournal%5D)">An analysis of the association of gastroenteric lesions with chronic wasting syndrome of marmosets.</a> Vet Pathol Suppl. 7, 141-162 (1982).</li>
<li>Logan, A. C., Khan, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+pathologic+changes+in+two+marmosets+with+wasting+syndrome%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Clinical pathologic changes in two marmosets with wasting syndrome.</a> Toxicol Pathol. 24 (6), 707-709 (1996).</li>
<li>Ludlage, E., Mansfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+care+and+diseases+of+the+common+marmoset+%28Callithrix+jacchus%29%5BTitle%5D)+AND+%22Comp+Med%22%5BJournal%5D)">Clinical care and diseases of the common marmoset (Callithrix jacchus).</a> Comp Med. 53 (4), 369-382 (2003).</li>
<li>Ross, C. N., Davis, K., Dobek, G., Tardif, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging+phenotypes+of+common+marmosets+%28Callithrix+jacchus%29%5BTitle%5D)+AND+%22J+Aging+Res%22%5BJournal%5D)">Aging phenotypes of common marmosets (Callithrix jacchus).</a> J Aging Res. 2012, 567143(2012).</li>
<li>Yoshimoto, T., Niimi, K., Takahashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curative+effects+of+tranexamic+acid+with+supportive+measures+on+wasting+marmoset+syndrome%5BTitle%5D)+AND+%22Comp+Med%22%5BJournal%5D)">Curative effects of tranexamic acid with supportive measures on wasting marmoset syndrome.</a> Comp Med. 66 (6), 1-6 (2016).</li>
<li>Niimi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+the+%CE%B11-proteinase+inhibitor+%28%CE%B11-antitrypsin%29+of+common+marmoset+and+intestinal+protein+loss+in+wasting+syndrome%5BTitle%5D)+AND+%22Biosci+Rep%22%5BJournal%5D)">Measurement of the α1-proteinase inhibitor (α1-antitrypsin) of common marmoset and intestinal protein loss in wasting syndrome.</a> Biosci Rep. 39 (7), 20190562(2019).</li>
<li>Otovic, P., Smith, S., Hutchinson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+glucocorticoids+in+marmoset+wasting+syndrome%5BTitle%5D)+AND+%22J+Med+Primatol%22%5BJournal%5D)">The use of glucocorticoids in marmoset wasting syndrome.</a> J Med Primatol. 44 (2), 53-59 (2015).</li>
<li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Administration+of+substances+to+laboratory+animals%3A+routes+of+administration+and+factors+to+consider%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li>
<li>Lewis, S. M., Hotchkiss, C. E., Ullrey, D. E. Chapter 13. Nutrition and nutritional diseases. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLewis%2C+SM+%22The+Laboratory+Primates%22">The Laboratory Primates</a>. Wolfe-Coote, S. , 181-208 (2005).</li>
</ol>

The authors have no conflicts of interest to disclose.

In 2016, we reported that tranexamic acid with supportive measures was an effective treatment for WMS, which was the first report to demonstrate WMS therapy without the use of glucocorticoids. In humans, glucocorticoids are considered the most effective treatment of IBD. However, predonizolone, one of the most commonly used glucocorticoids, is not suitable for WMS treatment because of its adverse effects. Although budesonide, a glucocorticoid, was reported for WMS treatment9, the therapy was relatively ineffective in animals with acute forms of WMS. Tranexamic acid is a plasmin inhibitor that has anti-inflammatory effects, and no notable side effects were seen in our original methods. However, although the original treatment protocol was definitely effective, it imposed a heavy load for animals and carers.
In the present study, the modified treatment methods for WMS were conducted to decrease both the animal's physiological load and the carers' workload. In the modified methods, the administration route of tranexamic acid was changed (from intraperitoneal to subcutaneous), which contributed to reducing the risk of injury to the abdominal organs. The tranexamic acid solution was not diluted in this protocol to reduce contamination risk and preparation time. In the original methods, 3.0 mL of the amino acid formulation were injected intravenously. However, the volume was somewhat large because 5.0 mL/kg is recommended to be administered as a bolus10. Therefore, the volume was reduced in the modified methods. 
The vitamin formulation used in this protocol contains vitamins B and C. In the original method, 0.5 mL of the vitamin formulation contains 2.5 mg of thiamine chloride hydrochloride, which was 2.5x the requirement of laboratory housed postweaning nonhuman primates11. In the present method, 0.1 mL of the vitamin formulation was administered, which contains 1.0 mg of thiamine chloride hydrochloride. The frequency of administration of the iron formulation and tranexamic acid solution was reduced in the modified methods, which contributed to the reduction of burden for the animals as well as the carers. 
As written in the Representative Results section, there were significant treatment effects in the modified methods. The average treatment term was 37.8 &plusmn; 25.34 days, which was shorter than that of the original methods (56 days). Since marmosets are sensitive to mental stress, prolonged treatments have the opposite effect on the animal such as reduced appetite. Thus, we recommend that the carers should comprehensively decide the timing of treatment termination based on not only the value of each parameter but also the animal's behavior.

Wasting marmoset syndrome (WMS) is a serious disease that is unique to captive common marmoset colonies. The main symptoms are weight loss, decreased muscle mass, anemia, hypoalbuminemia, and chronic enteritis1,2,3,4. Some studies have reported that 60.5% of euthanized captive marmosets had evidence of chronic enteritis5, and 31-44% of deaths involved this illness6. The high prevalence of this disease necessitated its effective treatment.
In 2016, we reported that tranexamic acid with supportive measures was an effective treatment for WMS7. In addition, intestinal protein loss of WMS-affected marmosets was significantly attenuated with this treatment8. Although the original treatment protocol was definitely effective, it included a somewhat high volume of intravenous injection, a high dose of vitamin formulation, and daily restraining. Since marmosets are susceptible to hand-capture and restraining, these actions must be less frequent. Thus, we aimed to reduce the heavy load for animals with some modification.
In the present paper, we will provide the modified methods: subcutaneous injection of undiluted tranexamic acid 5 times per week (instead of intraperitoneal injection of 5-fold-diluted tranexamic acid solution 7 times per week), 2.0 mL of amino acid formulation for intravenous injection (instead of 3.0 mL), 0.1 mL of vitamin formulation for subcutaneous injection (instead of 0.5 mL), and oral administration of iron formulation 5 times per week (instead of 7 times per week).

<table><tbody><tr><td>1.0 mL Syringe</td><td>Terumo Corporation, Tokyo, Japan</td><td>SS-01T</td><td>For subcutaneously injection of Tranexamic acid</td></tr><tr><td>10 mL Syringe</td><td>Terumo Corporation, Tokyo, Japan</td><td>SS-10SZ</td><td>For Intravenous Administration of Amino acid formulation</td></tr><tr><td>26 G Needle</td><td>Terumo Corporation, Tokyo, Japan</td><td>NN-2613S</td><td>For subcutaneously injection</td></tr><tr><td>27 G Butterfly Needle Type D</td><td>Terumo Corporation, Tokyo, Japan</td><td>SV-27DL</td><td>For Intravenous Administration of Amino acid formulation</td></tr><tr><td>5 mL Syringe</td><td>Terumo Corporation, Tokyo, Japan</td><td>SS-05SZ</td><td>For subcutaneously injection of Ringer&#039;s lactate and Vitamin formulation</td></tr><tr><td>Aminoleban Injection</td><td>Otsuka Pharma Factory Inc., Tokyo, Japan</td><td>1942</td><td>For subcutaneously injection of Ringer&#039;s lactate and Vitamin formulation</td></tr><tr><td>C-PARA</td><td>Takata Pharma Co., Ltd., Saitama, Japan</td><td>120-31960-0</td><td>Vitamin formulation</td></tr><tr><td>FCV Liquid</td><td>Minerva Corporation, Saitama, Japan</td><td></td><td>Iron formulation</td></tr><tr><td>Lactec Injection</td><td>Otsuka Pharma Factory Inc., Tokyo, Japan</td><td>0714</td><td>Ringer&amp;rsquo;s lactate&amp;nbsp;</td></tr><tr><td>Vasolamin Injection</td><td>Meiji Seika Pharma Co., Ltd., Tokyo, Japan</td><td>DBASOS</td><td>Tranexamic acid</td></tr></tbody></table>

modification of the treatment methods for wasting marmoset syndrome with tranexamic acid and supportive measures

Wasting marmoset syndrome (WMS), a serious disease in captive common marmoset (Callithrix jacchus) colonies, is associated with a high mortality rate. The specific cause of WMS is still unclear and there are few effective treatments. Previously, we had reported a tranexamic acid therapy with supportive measures as a useful treatment for WMS. In the present study, we describe the modified method: a combination of 0.1 mL of 5% tranexamic acid subcutaneously five times per week, 2.0 mL of amino acid formulation intravenously three times per week, 5.0 mL of Ringer's lactate with 0.1 mL of a vitamin formulation subcutaneously three times per week, and oral administration of 0.1 mL of an iron formulation five times per week. We also describe how to administer the solution intravenously via the saphenous vein with a tip of restraining the animal, as well as the detailed methods for oral and subcutaneous administration. The modified methods have comparable efficiency to the original WMS treatment method.

The modified treatment methods resulted in increased hematocrit (Figure 1A), serum albumin (Figure 1B), and body weight (Figure 1C) in the WMS-affected marmosets. Significant differences in hematocrit (P &#60; 0.05, Wilcoxon matched-pairs signed rank test), serum albumin (P &#60; 0.05, Wilcoxon matched-pairs signed rank test), and body weight (P &#60; 0.05, Wilcoxon matched-pairs signed rank test) were seen between pre and post treatment.
The treatment effects on the appearance were also observed. Figure 2 shows the appearance of a WMS-affected marmoset. Before treatment, tabefaction, arched back, rough fur (Figure 2A), and alopecia (Figure 2B) were observed, whereas no abnormal appearance was seen after treatment (Figure 2C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66768/66768fig01.jpg" /> 
Figure 1: Changes (pre- vs post-treatment) in hematocrit, serum albumin, and body weight in a marmoset receiving the modified treatment. (A) Hematocrit values, (B) serum albumin values, and (C) body weight. The dashed lines indicate the criteria values. Abbreviations: Htc = hematocrit; Alb = serum albumin; BW = body weight. <a href="https://www.jove.com/files/ftp_upload/66768/66768fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66768/66768fig02.jpg" /> 
Figure 2: Representative appearance of WMS-affected marmoset. (A) Appearance of pretreatment animal: tabefaction, arched back, and rough fur are observed. (B) Tail of pretreatment animal: alopecia is observed. (C) Appearance of posttreatment animal. (D) Tail of posttreatment animal. Abbreviation: WMS = wasting marmoset syndrome. <a href="https://www.jove.com/files/ftp_upload/66768/66768fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Developing a Safer and More Efficient Treatment Protocol for Wasting Marmoset Syndrome (WMS)

Here, we describe the modified treatment methods for wasting marmoset syndrome (WMS, also known as inflammatory bowel disease (IBD)-like disease) with tranexamic acid. We also present how to administer the therapeutic agents orally, subcutaneously, and intravenously.

Modification of the Treatment Methods for Wasting Marmoset Syndrome with Tranexamic Acid and Supportive Measures

Wasting marmoset syndrome (WMS), a serious disease in captive common marmoset (Callithrix jacchus) colonies, is associated with a high mortality rate. The ...

modification-treatment-methods-for-wasting-marmoset-syndrome-with

Research

JoVE Journal

Medicine

925 Views.  RIKEN Center for Brain Science. Here, we describe the modified treatment methods for wasting marmoset syndrome (WMS, also known as inflammatory bowel disease (IBD)-like disease) with tranexamic acid. We also present how to administer the therapeutic agents orally, subcutaneously, and intravenously.

Video: Author Spotlight: Developing a Safer and More Efficient Treatment Protocol for Wasting Marmoset Syndrome (WMS)

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Common Marmoset Model of Mother-Infant Intervention for Breastfeeding Disorders in the Presence of Paternal Inhibition and Maternal Neglect

Parents&#39; psychological stress during the perinatal and neonatal periods continues to increase in an environment of declining birthrates, aging populations, and shrinking family sizes. The increase in child abuse and neglect cases, most likely by inexperienced and insufficiently knowledgeable parents, necessitates education on childcare and intervention techniques in nursing and midwifery training. In particular, attachment formation early in life between mother and infant is crucial. To accurately teach sensitive and comprehensive information on intervention techniques for mother-child attachment formation, realistic videos, and educational materials are necessary. Although pseudoeducational materials are available, they might be limited in explaining complex realism, particularly to support breastfeeding that involves both parents and child and that encourages interaction between the two.
In a previous study in a common marmoset (Callithrix jacchus) model, we experimentally controlled infant feeding and nurturing through 24 h of constant sensing and collected 1 month of quantitative data on psychological indices that possibly translated to psychological development. Age-dependent dynamic visualization of these data by multivariate analyses inferred causal relationships between early parental feeding and psychobiological rhythm formation. In the same primate model, we identified a spontaneous case of breastfeeding failure in which the father inhibited his neonatal infant&#39;s feeding and the mother appeared to abandon nurturing, leading to clinically significant weight loss in the infant.
Thus, we explored intervention techniques to promote mother-infant interaction. The mother was trained to allow the infant to spontaneously explore her breast. Initially, the mother refused to display the feeding pose potentially due to pain associated with breast engorgement. Massage was used to soften the breast and feeding was reintroduced. We hypothesize that activation of instinctive attachment formation mechanisms by encouraging spontaneity in each parent and child is the key to successful feeding intervention.

Here, we present a nonhuman primate model of mother-infant intervention for breastfeeding disorders in the presence of paternal inhibition and maternal neglect. The mate model video complements education to support primate and human caregivers with respect to infants with breastfeeding problems such as pain.

Parents&#39; psychological stress during the perinatal and neonatal periods continues to increase in an environment of declining birthrates, aging ...

Behavior

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies.
Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

Immunology and Infection

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

Neuroscience

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

The Mouse Stroke Unit Protocol with Standardized Neurological Scoring for Translational Mouse Stroke Studies

The filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with intravascular reperfusion/recanalization. However, it lacks alignment with current clinical advances for stroke care (e.g., Stroke Units), usually employs subjective or vague neurological scoring among laboratories, and exhibits high acute-phase mortality. Here, we address these limitations with validated video-guided protocols. We present the mouse Stroke Unit (mSU) protocol with instructional videos and a decision algorithm (Risk Stratification Score), bridging the gap between clinical and mouse stroke modeling. To increase accuracy and sensitivity of stroke neurological scoring, we present for the first time a video-standardized format of the focal Experimental Stroke Scale (fESS) and prove its value up to 6 months post-stroke. Additionally, protocols for mice Ladder-rung test, as well as the known Cylinder test, for unbiased, quantitative assessment of limbs&#180; motor function are presented. Results highlight mSU&#39;s translational efficacy. Focal ESS (fESS) excels over other known scales in detecting focal stroke deficits, capturing recovery, and maintaining sensitivity for up to 6 months post-stroke. Ladder-rung and Cylinder tests objectively quantify and monitor fore- and hind-limb motor deficits, long-term. In summary, integrating mSU, fESS, and motor function tests provides a robust framework for clinically relevant stroke investigations. Our protocols improve the translational value in mouse stroke research.

This article presents a protocol for executing the highly translational and independently proven mouse Stroke Unit (mSU) concept in large-stroke mice. It also provides a standardized protocol for executing the focal experimental stroke scale in mice, which evaluates stroke focal deficits detected even long-term.

The filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with ...

Enhancing Survival and Development of Marmosets Using a Hand-Rearing Approach

Hand-Rearing Method for Infant Marmosets

The common marmoset (Callithrix jacchus) is a small and highly social New World monkey with high reproduction rates, which has been proven to be a compelling non-human primate model for biomedical and neuroscience research. Some females give birth to triplets; however, the parents cannot raise all of them. To save these infants, we have developed a hand-rearing method for raising newborn marmosets. In this protocol, we describe the formula of the food, the time for feeding, the configuration of the temperature and humidity, as well as the adaptation of the hand-reared infants to the colony environment. This hand-rearing method significantly increases the survival rate of marmoset infants (without hand-rearing: 45%; with hand-rearing: 86%) and provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. As the method is practical and easy to use, we anticipate that it could also be applied to other labs working with common marmosets.

Author Spotlight: Marmoset Research - Scope and Challenges 

Here, we describe a hand-rearing method for raising infant marmosets in an animal incubator. This method greatly increases the survival rate of marmoset infants, which provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments.