Combined abdominal heterotopic heart and aorta transplant model in mice.

BACKGROUND: Allograft vasculopathy (AV) remains a major obstacle to long-term allograft survival. While the mouse aortic transplantation model has been proven as a useful tool for study of the pathogenesis of AV, simultaneous transplantation of the aorta alongside the transplantation of another organ may reveal more clinically relevant mechanisms that contribute to the pathogenesis of chronic allograft rejection. Therefore, we developed a combined abdominal heart and aorta transplantation model in mice which benefits from reducing animal and drug utilization, while providing an improved model to study the progressive nature of AV.
METHODS: The middle of the infrarenal aorta of the recipient mouse was ligatured between the renal artery and its bifurcation. Proximal and distal aortotomies were performed at this site above and below the ligature, respectively, for the subsequent anastomoses of the donor aorta and heart grafts to the recipient infrarenal aorta in an end-to-side fashion. The distal anastomotic site of the recipient infrarenal aorta was connected with the outlet of the donor aorta. Uniquely, the proximal anastomotic site on the recipient infrarenal aorta was shared to connect with both the inlet of the donor aorta and the inflow tract to the donor heart. The outflow tract from the donor heart was connected to the recipient inferior vena cava (IVC).
RESULTS: The median times for harvesting the heart graft, aorta graft, recipient preparation and anastomosis were 11.5, 8.0, 9.0 and 40.5 min, respectively, resulting in a total median ischemic time of 70 min. The surgery survival rate was more than 96% (29/30). Both the syngeneic C57Bl/6 aorta and heart grafts survived more than 90 days in 29 C57Bl/6 recipients. Further, Balb/c to C57Bl/6 allografts treated with anti-CD40L and CTLA4.Ig survived more than 90 days with a 100% (3/3) survival rate. (3/3).
CONCLUSIONS: This model is presented as a new tool for researchers to investigate transplant immunology and assess immunosuppressive strategies. It is possible to share a common anastomotic stoma on the recipient abdominal aorta to reconstruct both the aorta graft entrance and heart graft inflow tract. This allows for the study of allogeneic effects on both the aorta and heart from the same animal in a single survival surgery.

Introduction

Solid organ transplantation is one of the only viable interventions for patients with end-stage organ failure. Beyond acute rejection, which is most prevalent in the first year post-transplantation and can be actively managed with adjustments in immunosuppressive regimen or induction therapy [1, 2], the long term survival of transplanted hearts is impeded by graft failure, malignancy, cardiac allograft vasculopathy (CAV) and renal failure [1, 2]. Chronic allograft rejection remains one of the leading causes of graft failure one year post-transplantation [1, 2].

It is well known that T cell-mediated immune responses play a central role in acute allograft rejection [3]. Current immunosuppressive regimens targeting T cell activation and effector cell function have led to dramatic reductions in acute rejection. However, chronic allograft injury leading to graft failure and CAV remains a major obstacle to the long-term allograft survival [1, 2, 4]. Although the exact etiology remains unclear, multifactorial mechanisms including both immunological and non-immunological components contribute to the development of chronic allograft rejection [5, 6]. In the heart, chronic allograft rejection presents as CAV, and is characterized as an accelerated form of atherosclerosis which occurs in the arteries of the transplanted heart [5, 6]. CAV is initiated by a combination of ischemia/reperfusion injury and alloimmune injury which results in endothelial dysfunction [5, 6]. This leads to a progressive fibroproliferative disease with intimal smooth muscle cell proliferation leading to progressive vessel occlusion, thrombotic events and eventual graft failure [5, 6]. Allograft vasculopathy (AV) can also occur in a number of solid organ transplant settings (i.e. lung, kidney, etc.) with similar histopathological characteristics to CAV [7]. Despite current immunosuppressive regimens, CAV is reported in almost 50% of patients 10 years post transplantation [1, 2]. As such, there is a growing need for the development of reliable animal models to decipher underlying mechanism of CAV and further optimize and develop therapeutic strategies to address this major health burden.

Heterotopic heart transplantation in mice has been considered the pre-eminent model to study transplant immunology since it was introduced by Corry and Russell in 1973 [8]. Without immunosuppression, transplantation of a fully MHC-mismatched cardiac allograft induces strong alloreactive T cell responses that mediate rapid graft rejection [9]. In this regard, the heterotopic heart transplant model in mice recapitulates the pathological process of acute allograft rejection. To study chronic allograft injury, immunosuppressive drugs have used in this model to suppress the acute immune response. However, the majority of currently used immunosuppressive drugs already target T cell activation to prevent acute transplant rejection. Thus, the development of AV is likely a reflection of sub-acute immunological events. Further, it has been suggested that these immunosuppressive agents may also contribute to the development of AV [10].

The aortic transplant model in mice has been used to study some components of the immunological and/or molecular mechanisms of AV. However, fully MHC-mismatched aortic allografts demonstrate long-term survival even in absence of immunosuppression [11]. On one hand, this allows for the study of mechanisms that contribute to AV in the absence of a strict influence from acute rejection, as acute rejection episodes have long been considered as a risk factor for the future development of AV [12]. However, as aortic allografts do not undergo acute rejection, it remains controversial whether the vascular changes observed in aortic allografts accurately represent those that occur in solid organ transplants [13].

To address some of these issues, investigators have developed combined heart and aorta/carotid artery transplantation models in mice to investigate the potential impact of acute rejection on CAV [13, 14]. As expected, compared with isolated carotid allografts, a significantly more intimal hyperplasia of carotid allografts was noted in aorta transplanted in combination with a heart graft, indicating that CAV is promoted by acute parenchymal rejection of the heart [14]. This observation has further highlighted the need for transplantation of a parenchymal organ in combination with an aortic graft to more accurately model the events which contribute to the pathogenesis of CAV in human heart allografts. Despite impressive results made utilizing their models, the transplantation of heart and aorta/carotid graft into two different operative sites (abdominal and cervical respectively) causes increased operative traumas and prolonged operative times, limiting its widespread use. In order to simplify the surgical protocol and remove the need for multiple survival surgeries (resulting in reductions in graft ischemia and overall operative times), we developed a new technique to transplant heart and aorta graft in a single site within the abdomen. Further, transplantation of the aorta and heart in a single surgery reduces donor animal utilization. The aim of our study was therefore to evaluate a new microsurgical technique of simultaneous heterotopic abdominal heart and aorta transplantation in mice, as well as to assess the feasibility of sharing a common anastomotic stoma on the recipient infrarenal aorta to reconstruct both the aorta-graft entrance and heart-graft inflow tract.

Our results demonstrate that this new surgical protocol is reliable, reproducible and improves upon existing techniques. Further studies using this model will provide insight into the clinical process of chronic allograft rejection and a useful tool for assessing the novel immunosuppressive strategies for its prevention. In addition, to our knowledge, this is the first demonstration of a novel microsurgical technique in which an anastomotic stoma on the recipient infrarenal aorta can be connected with two individual vessels, providing a new pathway of revascularization for multi-organ transplantation.

Materials and methods

Animals

Adult female Balb/c mice 6–10 weeks of age (20-25g body weight) and male C57Bl/6 mice, 6–10 weeks of age (20-25g body weight) were purchased from Jackson Laboratory (Bar Harbor, MD, USA). Animals were housed under standard conditions subjected to regular 12-hour light-dark cycles. Water and chow were supplied ad libitum. Animal experiments were conducted in accordance with an approved Washington University School of Medicine (WUSM) Institutional Animal Care and Use Committee (IACUC) protocol (#20190173). Anesthesia was induced by a mixture of ketamine (80–100 mg/kg)/xylazine HCl (8–12 mg/kg), intraperitoneally (i.p.) and maintained with 1–2% isoflurane gas, as required.

Donor operation

A longitudinal laparotomy was made, and abdominal contents were reflected to the left side to expose the inferior vena cava (IVC). Approximately 1.0 mL of cold saline containing heparin (100 U/mL) was injected into the IVC. After 1 minute for systemic heparinization, an aortotomy of the abdominal aorta was made to decompress the blood circulatory system. Subsequently, a bilateral thoracotomy was performed through the ribs along both sides of the thoracic spine, then the anterior chest wall was levered up cranially. The IVC around diaphragmatic hiatus was clamped with a hemostat, above that, the IVC was cannulated and then 0.5 mL of cold heparin (100 U/mL) is again infused into the right atrium. The thymus was resected to expose the aortic arch and pulmonary artery. The ascending aorta was transected proximal to the innominate artery. The pulmonary artery was transected proximal to its bifurcation. The IVC and the right superior vena cava (SVC) were proximally ligated with 7–0 sutures. A 5–0 ligature was placed underneath the IVC and the right SVC and around the heart to ligate all other vessels en bloc including the pulmonary veins and the left SVC as distally as possible. The IVC, the right SVC and the remaining connective tissues were all dissected distally in order. The donor heart was then gently detached from the thorax and stored in ice-cold saline. In the following steps, the donor thoracic descending aorta would be harvested as described by Cho et al [15]. Lungs were resected, then the diaphragm was divided as close to the aorta as possible to expose descending aorta. The descending aorta was further flushed by cold heparin (100 U/mL) solution from the aorta bellow the diaphragm. The parietal pleura in front of the descending aorta were divided. Intercostal arteries were carefully divided from the segment of the descending aorta between the left subclavian artery proximally and the diaphragmatic hiatus distally. The thoracic descending aorta was harvested and stored in ice-cold saline. Although the thoracic aorta can be divided into three segments for individual transplantation, it is preferable to use the proximal section, as it is most compatible in diameter with ascending aorta of the donor heart, and therefore facilitates the subsequent anastomosis.

Recipient operation

A midline laparotomy was made from the pubis to the xiphoid, then a micro-retractor was placed to expose the abdominal cavity. The intestines were gently retracted right outside the abdomen and covered with moistened gauze and kept moist throughout the procedure with saline. Using two cotton swabs, the abdominal aorta and the IVC below the renal vessels and above its bifurcation were exposed and dissected gently from the surrounding tissues. Of note, a complete isolation of infrarenal aorta from IVC was unnecessary for performing subsequent end-to-side anastomosis. One or two groups of the lumber arteries and veins underneath the abdominal aorta and IVC were ligated with 9–0 silk sutures. To interrupt the blood flow in both the aorta and the IVC, a 5–0 silk was single-tied at the proximal side of its bifurcation to allow it to be untied easily after anastomosis, and then a micro clamp was placed just at below the renal vessels. Next, the middle of the sequestered aorta was sutured with a 9–0 nylon suture (). Thus, the aorta was partitioned into proximal and distal anastomotic areas without transection. The proximal and distal aortotomies were performed above and below the ligature respectively, for the subsequent anastomoses of the aorta- graft into the recipient in an end-to-side fashion (). We preferred to make the aortotomy by a simple and reliable technique described by Mao et al. [16]. Briefly, the tip of a 4 mm (3/8) needle was longitudinally passed in and slightly out of the anterior wall of the recipient aorta and kept in position by a needle holder. By gently lifting the needle upwardly, the piece of the anterior wall of the aorta above the needle could be easily excised by cutting underneath the needle with fine scissors, so that an approximate 1 mm elliptical opening with the trim-edge was made. In the same way, a venotomy in parallel with proximal aortotomy was made for the subsequent construction of heart-graft outflow tract. The openings of the aorta and the IVC were flushed with saline to remove the blood.

Diagrammatic (A) and photographic (B) representation of the middle of the recipient’s infrarenal aorta between renal artery and its bifurcation, which was ligatured with a 9–0 nylon suture.

(A) Diagrammatic representation of the proximal and the distal aortotomies on the recipient’s infrarenal aorta were performed above and below the ligature, respectively, for the subsequent anastomoses of the donor’s aorta graft into the recipient in an end-to-side fashion. (B) Diagrammatic representation of the left side of the anastomotic site of the recipient’s infrarenal aorta, which was anastomosed to the posterior wall of the donor’s aorta graft within the vessel.

The aorta graft was placed on the left side of the recipient abdomen, and then covered with gauzes moistened with icy cold saline. First, using 11–0 nylon surgical suture, the aorta-graft exit was constructed in the distal anastomotic site of the recipient infrarenal aorta in an end-to-side fashion. The posterior and the anterior walls of the anastomosis were completed inside and outside the vessels with 4–5 continuous running sutures, respectively. The aorta-graft entrance and heart-graft inflow tract were then constructed by sharing to connect the proximal anastomotic site of the recipient infrarenal aorta. Briefly, the left side of the anastomotic site of the recipient infrarenal aorta was anastomosed to the posterior wall of the donor aorta-graft inside vessel (). In the following step, the donor heart graft was placed on the left side of the recipient abdomen with the remnant of the ascending aorta underneath the pulmonary artery and perpendicular to the clamped vessels, and then covered with gauze moistened with ice-cold saline. The anterior wall of the aorta-graft was anastomosed to the posterior wall of the ascending aorta of the heart-graft inside vessel (). The anterior wall of the ascending aorta of the donor heart was then anastomosed to the right side of the anastomotic site of the recipient infrarenal aorta outside vessel (). The last step in the procedure was to construct the heart-graft outflow tract, similarly, by an end-to-side anastomosis between the donor pulmonary artery and the recipient IVC with continuous running sutures of the posterior wall inside vessel and the anterior wall outside vessel ().

Diagrammatic (A) and photographic (B) representation of the anterior wall of the aorta graft, which was anastomosed to the posterior wall of the ascending aorta of the heart graft within the vessel.

Diagrammatic (A) and photographic (B) representation of the anterior wall of the ascending aorta of the heart graft, which was anastomosed to the right side of the anastomotic site of the recipient’s infrarenal aorta.

Diagrammatic (A) and photographic (B) representation of the outflow tract (pulmonary artery) of the donor’s heart graft, which was connected to the recipient’s inferior vena cava (IVC).

After completion of anastomoses, the distal ligature was untied first, followed by removal of the proximal clamp. The anastomotic sites were gently pressed by two cotton swabs for several seconds, to minimize/stop bleeding. Optionally, small pieces of hemostatic agent, spongostan, can be placed around the anastomotic sites to prevent anastomotic bleeding. Normally, the aorta- and heart- graft immediately fills with blood and consequently becomes bright red in color. Prominent pulsations of the aorta-graft were visible and after a short episode of fibrillation, sinus rhythm resumed in the heart-graft (). After ensuring there was no further bleeding, the abdominal incision was closed with 4–0 suture by continuous running stitches.

Immunosuppression

For a subset of additional transplants, the long-term viability of this combined aorta and heart transplant model were tested in syngraft (C57Bl/6 to C57Bl/6) (n = 3) and allograft (Balb/c to C57Bl/6) models (n = 3), with the administration of 250 μg of InVivoMab anti-mouse CD40L (CD154; clone MR-1; Bio X Cell, West Lebanon, NH) on the day of transplantation, and 200 μg of InVivoMab recombinant CTLA-4-Ig (hum/hum; Bio X Cell) on day 2 post-transplantation [17, 18].

Histology and imaging

Four representative sections from each aortic and heart graft were stained with hematoxylin and eosin (H&E; Leica Biosystems, Buffalo Grove, IL) or Movat’s pentachrome (Newcomer Supply, Middleton, WI) and digital images were captured using an Olympus BX61 microscope (Center Valley, PA).

Results

A total of 30 combined heterotopic abdominal heart and aorta syngrafts were performed using this technique in C57Bl/6 mice. A median time of 11.5 min (10–14 min range) and 8.0 min (7–10 min range) was required for harvesting of the donor heart and aorta grafts, respectively. Preparation of the transplant recipient required a median time of 9.0 min (8–11 min range), and anastomoses of the donor organs required a median time of 40.5 min (38–44 min range). There was a median total ischemic time of 70 min (65–74 min range) (Fig 6). The surgery survival rate of syngrafts was greater than 96% (29/30). One mouse died due to anastomotic hemorrhage on the same day as transplantation. The remaining 29 of 30 syngrafts survived greater than 90 days with maintained transplanted heart function (as assessed by palpation).

Fig Median time for donor heart harvest (red), donor aorta harvest (blue), transplant recipient preparation (green), total anastomosis time for the combined transplant procedure (pink) and total ischemic time (orange). Data are represented as median time (in minutes) ± the range in time for all procedures. n = 30 transplants.

To validate this surgical method, we subsequently performed allografts using Balb/c donor aorta and hearts transplanted into C57Bl/6 recipients (n = 3) in the presence of double co-stimulatory blockade [17, 18] or syngrafts using C57Bl/6 donor aorta and hearts transplanted into C57Bl/6 recipients (n = 3) as a control. For this group, 3/3 allografts and 3/3 syngrafts survived for more than 90 days, with maintained transplanted heart function (as assessed by palpation). Histological examination of aorta (Fig 7A) or cardiac (Fig 7B) grafts revealed long-term survival with a mild immune response, due to the strong immunosuppressive effect of double co-stimulatory blockade.

ig Representative micrographs of the aortic (A) and cardiac (B) grafts 90+ days post-transplantation treated with anti-CD40L and CTLA4.Ig stained with hematoxylin & eosin. Scale bars = 100 μm for (A); 500 μm for (B).

Discussion

AV is resistant to current immunosuppressive therapies and a major obstacle to long-term survival in solid organ transplantation. Although, there are controversies concerning the relative contribution of acute transplant rejection to the subsequent development of AV, current evidence increasingly suggests that acute rejection is a strong risk factor for the late development of AV [1, 2]. Indeed, clinical reduction in acute rejections has significantly decreased the incidence of chronic rejection graft failure [1, 2, 19].

The aortic transplant model in mice is a useful tool for studying AV, based on the formation of consistent lesions which can be easily quantified [20]. However, unlike solid organ allografts, isolated aortic allografts can achieve long-term acceptance in the absence of immunosuppressive interventions, sparing them from the influence of acute rejection. Therefore, this model lacks the contribution of cellular and soluble factors present in acute rejection that may augment the pathogenesis and evolution of CAV. In contrast, the strategy of combining aortic transplantation with solid organ transplantation includes the influence of systemic factors related to acute allograft rejection on the development of CAV, thus providing a model that may more accurately represent chronic allograft rejection. Indeed, Soleimani et al. demonstrated a marked increase in neointimal area in C57Bl/10 carotid grafts co-transplanted with Balb/c cardiac allografts into a C3H recipient mouse, compared to C57Bl/10 carotid grafts into C3H recipients [14]. This observation confirmed that the acute parenchymal rejection is an important contributor to CAV, and further emphasizes that a combined model may serve as a more clinically relevant model to investigate the mechanisms involved in the development of AV.

Researchers have developed the combined aorta/carotid and heart transplantation models in mice, in which the aorta/carotid and heart are transplanted into two different surgical sites (abdomen and cervical region, respectively). A likely rationale for the use of an additional surgical site is that there is insufficient space along the recipient abdominal aorta between the renal artery and its bifurcation to make three aortotomies for connecting aorta-graft entrance and exit as well as heart-graft inflow tract. Thus, a cervical operation has to be performed to accommodate another graft. However, two surgical sites of operations are associated with additional surgical procedures and prolonged anaesthesia, frequently resulting in increased surgical complications and mortality. To avoid the unnecessary surgical traumas and shorten the ischemia and overall operative times, we developed a simplified model, in which two individual grafts can be accommodated in a single surgical site of abdomen with a high success rate.

Several arterial transplantation models have been well-established in mice [20-24]. Both aortic and carotid grafts are most commonly used for study of CAV [21, 22]. Compared to carotid segments with similar length, aortic grafts are larger in size, and therefore can provide more tissue for subsequent histological and biochemical analysis. In our experience, a whole-length of thoracic ascending aorta from a single donor is long enough to be divided and transplanted into three to four individual recipients (unpublished observation). Therefore, use of an aortic graft rather than a carotid graft is superior in such models.

In mice, aortic grafts can be anastomosed to the recipient infrarenal aorta by either an end-to-end [20] or an end-to-side [11] anastomosis pattern. Due to the obvious disparity in size between the thoracic and abdominal segments of the aorta, the end-to-end pattern is technically more difficult, with a higher incidence of anastomotic complications (up to 20% of thrombotic complications reported) [20]. In contrast, the end-to-side pattern is technically more facile, leading to a greater than 98% success rate [11]. In addition, a mild dissection of the infrarenal aorta from IVC is sufficient to perform an end-to-side anastomosis. Thus, the donor operation is simplified by obviating the dissection of abdominal vessels, which are very easily injured. Of note, a potential limitation of this pattern is that a curved loop of aortic graft may alter blood flow patterns and produce unexpected and unreliable experimental results. To address this issue, after completion of the procedure of end-to-side anastomosis, Sun et al. [11] tried to convert an end-to-side to a quasi end-to-end anastomosis by transection of the native infrarenal aorta between the anastomotic sites of aortic allograft entrance and exit. However, this technique appeared to increase the likelihood of kinking at the anastomotic sites [25]. Importantly, data showed a loop and an interposition of aortic allograft made by end-to-side and end-to-end pattern respectively, yielded similar experimental results [25]. Therefore, on the basis of merit, we combined the end-to-side anastomosis pattern of aortic transplant without transection of native infrarenal aorta described by Cho et al. [15] for our study.

We used the abdominal site to implant both heart and aortic allografts, because the abdominal infrarenal aorta is much larger in size and longer than the carotid artery. Although the infrarenal aorta was divided from the IVC, a complete separation of these vessels from each other was unnecessary. Instead, a mild dissection of the abdominal vessels was easily performed and adequate to facilitate an end-to-side anastomosis. In order to transplant both heart and aorta grafts, three anastomotic stomas in the recipient infrarenal aorta were needed for reconstruction of aorta-graft entrance and exit as well as heart-graft inflow tract. However, we discovered that two adequate aortotomies, at a maximum, can be made in recipient infrarenal aorta for anastomosis, despite attempting to replace the distal clamp with a ligature of 5–0 silk to maximize the usage of the infrarenal aorta. To address this issue, we adopted the proximal anastomotic stoma on the recipient infrarenal aorta to connect both aorta-graft entrance and heart-graft inflow tract, increasing the available space along the recipient infrarenal aorta. Results obtained from our study demonstrated this novel microsurgical technique can be easily performed with a high success rate, without thrombotic occlusion or stenosis observed in heart and aortic allografts. This novel technique also provides a new pathway in revascularization for multi-organ transplantation within a limited space.

Su et al. [26] argued that an entire everting suture technique was able to decrease the anastomotic complications. However, in our laboratory, we favored the parachute technique to suture the vessel posterior walls inside the vessels (inverting suture) and anterior walls outsider the vessels (everting suture). In this way, grafts did not need to be re-positioned to the opposite side, and therefore the possibility of increased surgical complexities and warm ischemia times was further limited. In our previous experience with this method, a success rate of greater than 90% could be reliably achieved by a skilled microsurgeon for heterotopic heart transplantation in mice. In our current model, we adopted the same suture technique and achieved a similarly high success rate. In addition, using a technique described by Mao et al. to create the aortotomy and the venotomy [16], an elliptical anastomotic stoma can be easily made with a trim-edge, which may facilitate the vascular anastomosis. Nevertheless, proficiency in microsurgical techniques is required to enable researchers to achieve reliable and reproducible results with this model.

Conclusions

In conclusion, our study presents a simple and reliable technique for combined heterotopic abdominal heart and aorta transplantation in mice, which can be achieved by sharing a common anastomotic stoma on the recipient infrarenal aorta with two individual graft vessels. Further studies using this model should provide additional insight into the mechanism of CAV and reveal correlation between the acute rejection and the subsequent development of CAV. In addition, the novel technique applied in this model provides a new pathway for revascularization and increases availability of methodological options for the development of experimental animal models.

(XLSX)

Click here for additional data file.

1 Apr 2020

PONE-D-20-06026

Combined abdominal heterotopic heart and aorta transplant model in mice

PLOS ONE

Dear Dr. Wong,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by May 16 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Robert Jeenchen Chen, MD, MPH

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.  Thank you for stating in your Funding Statement:

'This work is supported, in part, by the International Society for Heart and Lung

Transplantation (ISHLT) Joel D. Cooper Career Development Award (BWW).'

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

Additional Editor Comments (if provided):

Please address the issues and concerns from the reviewers.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: “Combined Abdominal Heterotopic Heart and Aorta Transplant Model in Mice” is a manuscript that presents a novel microsurgical technique intended to improve upon the heart and aorta/carotid graft. The presented technique improves on the existing one because it has shorter graft ischemic times and is centered in only one body cavity. This model and the existing model are important as it may be used to recapitulate the clinical process of aortic vasculopathy. The methodology of the surgery is meticulously explained, and the statistics are appropriately performed. The illustrations are of good quality and add to the overall work. The discussion content is well thought-out and provides context for the study. This paper is marred by poor grammar, and since PLOS one does not provide proofreading and copyediting support, I do not believe it is publishable in its current state. I also suggest the following minor revisions for this manuscript in order to consider it for publication:

It would be helpful to the reader to provide the true distributions of the times for the operation (ischemic time, harvesting time), instead of reporting the approximate times. The authors should present the median time and the distribution (this could be represented graphically as well).

The validation of the new surgical method should be more adequately described. The authors write, “we subsequently performed allografts using Balb/c donor aorta and hearts transplanted into C57Bl/6 recipients in the presence of double co-stimulatory blockade”. The number of replicates of this procedure should be detailed, and it should be noted that the histologic examination of the aorta and cardiac graphs (6a and 6b) are representative of these experiments, if that is in fact the case.

Please add citations for the following sentence:

• Chronic allograft rejection remains the leading cause of graft loss one-year post-transplantation.

PLOS one does not provide any copyediting and proofreading service. There are multiple grammatical errors found in this manuscript. I believe that it is not publishable in its current state. I would suggest proofreading the article again. Examples of the many grammatical errors found throughout the paper are listed below:

• The result has further heightened the need for a parenchymal organ in combination with aorta transplantation in exploring clinic(al) relevance of CAV.

• Our results demonstrate that this new model is characterized by the reliable reproducibility.

oShould this sentence include “the”?

• After 1 minute for the systemic heparinization, an aortotomy of abdominal aorta is made to decompress the blood circulatory system,

othis should be an aortotomy of “the” abdominal aorta

• The IVC and the right superior vena cava (SVC) were proximally ligated with 7-0 sutures, respectively.

oIn this case respectively does not make sense.

Reviewer #2: Dear authors,

Thank you for submitting your manuscript titled "Combined abdominal heterotopic heart and aorta transplant model in mice" to PLOS ONE. It was a very well written manuscript and I commend you on accomplishing this very difficult microsurgery. Your description of the operation, as well as the diagrams truly help the reader follow along the operation. I do have a couple questions :

1. Have you compared this technique to the "conventional" heterotopic heart transplantation in mice? As in, have you compared the histology, alloresponses, coronary inflammation, etc between the techniques. This technique seems more difficult, with more anastomoses. If everything is similar, is there a reason to do this technique?

2. Unlike other solid organs, the heart is a dynamic structure that can fail with improper preload and afterload management which is critical post-transplantation. This can affect immunosuppression. Since this technique creates an organ in series, it may not be representative of the pressures seen in a real transplant. This may not matter in a liver for example. Do you think that since the heart is not exposed the similar pressures as in an orthotopic heart transplant that this technique may not be the best to address CAV? Please address. I think this is a major limitation of such models.

3. Despite heparin, there could still be issues with thrombosis. How can you tell if the coronaries are patent in the transplanted heart? Is the mouse continued on anticoagulation?

4. Do the transplant hearts go into arrhythmia?

5. Please review grammar and syntax.

Thank you very much for a very interesting manuscript.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Apr 2020

Please find our response to the reviewers and editor comments in the attached 'Response to Reviewers' file.

Submitted filename: PONE-D-20-06026_Response to Reviewers.docx

Click here for additional data file.

19 May 2020

PONE-D-20-06026R1

Combined abdominal heterotopic heart and aorta transplant model in mice

PLOS ONE

Dear Dr. Wong,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Before we accept the manuscript, please address the issue found by one reviewer:

At the end of the first paragraph of the results the following sentence is hard to understand: “Survival of both the syngeneic aorta and heart graft was greater than 90 days.” Is this referring to all of the mice that survived the procedure?

I do not believe that the graft survival should be presented as 6/6 in the second paragraph of the results (since you are referring to three cardiac graphs and three aortic graphs). I think it should be presented as 3/3 cardiac graphs and 3/3 aortic graphs.

==============================

We would appreciate receiving your revised manuscript by Jul 03 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Robert Jeenchen Chen, MD, MPH

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors of Combined abdominal heterotopic heart and aorta transplant model in mice present a much improved study detailing a new microsurgical technique aimed at studying allograft vasculopathy. My prior comments on their manuscript have been adequately addressed; the grammar is much improved and the distributions of the procedure times are adequately detailed for the reader. The article’s introduction and discussion have been largely re-written, and the article is now fit for publication. Below, I have two comments for the authors to consider.

At the end of the first paragraph of the results the following sentence is hard to understand: “Survival of both the syngeneic aorta and heart graft was greater than 90 days.” Is this referring to all of the mice that survived the procedure?

I do not believe that the graft survival should be presented as 6/6 in the second paragraph of the results (since you are referring to three cardiac graphs and three aortic graphs). I think it should be presented as 3/3 cardiac graphs and 3/3 aortic graphs.

Reviewer #2: Dear authors,

Thank you for submitting your manuscript titled "Combined abdominal heterotopic heart and aorta transplant model in mice" to PLOS ONE. I would like to also thank you for the time and effort spent on the revisions. It is evident that significant portions of this manuscript has been revised. Grammatical errors have been addressed and the overall ease of reading has improved. Furthermore, you have addressed all of the comments submitted by myself and my co-reviewer in a very thorough manner. Please ensure grammar and syntax are appropriate. I would like to accept this manuscript.

Thanks

Reviewer #3: The methodology of the surgery is meticulously explained with nice schemes and photos. I have no further comment to be addressed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 May 2020

Please find response to the reviewer's comments in the attached file, and also copied and pasted below:

RESPONSE TO THE REVIEWER’S COMMENTS

At the end of the first paragraph of the results the following sentence is hard to understand: “Survival of both the syngeneic aorta and heart graft was greater than 90 days.” Is this referring to all of the mice that survived the procedure?

RESPONSE: The highlighted sentence indeed refers to all of the syngrafts that survived the surgical procedure (29 of the 30 performed in this group). We have revised the sentence in the manuscript to clarify this detail as follows:

“The remaining 29 of 30 syngrafts survived greater than 90 days with maintained transplanted heart function (as assessed by palpation).”

I do not believe that the graft survival should be presented as 6/6 in the second paragraph of the results (since you are referring to three cardiac graphs and three aortic graphs). I think it should be presented as 3/3 cardiac graphs and 3/3 aortic graphs.

RESPONSE: The previous sentence was meant to refer to 6 of 6 total transplants for this experimental group (three Balb/c to C57Bl/6 allografts receiving double co-stimulatory blockade and three C57Bl/6 to C57Bl/6 syngrafts). We have revised the sentence in the manuscript to clarify this detail as follows:

“For this group, 3/3 allografts and 3/3 syngrafts survived for more than 90 days, with maintained transplanted heart function (as assessed by palpation.”

Submitted filename: PONE-D-20-06026R2_Response to Reviewers.docx

Click here for additional data file.

5 Jun 2020

Combined abdominal heterotopic heart and aorta transplant model in mice

PONE-D-20-06026R2

Dear Dr. Wong,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Robert Jeenchen Chen, MD, MPH

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: No Further Comments. The authors have sufficiently revised the manuscript. It is now fit for publication in Plos One.

Reviewer #3: Authors revised the manuscript in accordance with previous reviewers' comments.

I have no further comment to be addressed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

10 Jun 2020

PONE-D-20-06026R2

Combined abdominal heterotopic heart and aorta transplant model in mice

Dear Dr. Wong:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Robert Jeenchen Chen

Academic Editor

PLOS ONE