Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

Florian S Frueh; Bijan Jelvani; Claudia Scheuer; Christina Körbel; Bong-Sung Kim; Pietro Giovanoli; Nicole Lindenblatt; Yves Harder; Emmanuel Ampofo; Michael D Menger; Matthias W Laschke

doi:10.1371/journal.pone.0239517

. 2020 Oct 6;15(10):e0239517. doi: 10.1371/journal.pone.0239517

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

Florian S Frueh ^1,^2,^*, Bijan Jelvani ¹, Claudia Scheuer ¹, Christina Körbel ¹, Bong-Sung Kim ², Pietro Giovanoli ², Nicole Lindenblatt ², Yves Harder ^3,⁴, Emmanuel Ampofo ¹, Michael D Menger ¹, Matthias W Laschke ¹

Editor: Zhejun Cai⁵

PMCID: PMC7537894 PMID: 33021999

Abstract

Vascularized lymph node (VLN) transfer is an emerging strategy to re-establish lymphatic drainage in chronic lymphedema. However, the biological processes underlying lymph node integration remain elusive. This study introduces an experimental approach facilitating the analysis of short-term molecular and cellular effects of ischemia/reperfusion on VLN flaps. Lymph node flaps were dissected pedicled on the lateral thoracic vessels in 44 Lewis rats. VLN flaps were exposed to 45 or 120 minutes ischemia by in situ clamping of the vascular pedicle with subsequent reperfusion for 24 hours. Flaps not exposed to ischemia/reperfusion served as controls. Lymph nodes and the perinodal adipose tissue were separately analyzed by Western blot for the expression of lymphangiogenic and angiogenic growth factors. Moreover, morphology, microvessel density, proliferation, apoptosis and immune cell infiltration of VLN flaps were further assessed by histology and immunohistochemistry. Ischemia for 120 minutes was associated with a markedly reduced cellularity of lymph nodes but not of the perinodal adipose tissue. In line with this, ischemic lymph nodes exhibited a significantly lower microvessel density and an increased expression of VEGF-D and VEGF-A. However, VEGF-C expression was not upregulated. In contrast, analyses of the perinodal adipose tissue revealed a more subtle decrease of microvessel density, while only the expression of VEGF-D was increased. Moreover, after 120 minutes ischemia, lymph nodes but not the perinodal adipose tissue exhibited significantly higher numbers of proliferating and apoptotic cells as well as infiltrated macrophages and neutrophilic granulocytes compared with non-ischemic flaps. Taken together, lymph nodes of VLN flaps are highly susceptible to ischemia/reperfusion injury. In contrast, the perinodal adipose tissue is less prone to ischemia/reperfusion injury.

Introduction

The lymphatic system is essential for tissue fluid homeostasis in higher vertebrates [1]. Furthermore, it is involved in the regulation of immunosurveillance and the absorption of dietary fats [2]. The lymphatic vasculature consists of capillaries, precollecting vessels and collecting lymphatic trunks, which form a complex network with interposed lymph nodes. Its disruption may result in lymphedema, a condition characterized by limb swelling, chronic interstitial inflammation and connective or adipose tissue deposition [3,4]. In developed countries, cancer-related lymphedema represents the most common form. It is estimated that ~ 1 out of 6 patients treated for a solid tumor develops lymphedema related to type and extent of treatment, anatomical location, and length of follow-up [5]. Particularly breast cancer is associated with high rates of secondary lymphedema. In fact, > 20% of cancer survivors undergoing axillary lymph node dissection develop arm lymphedema [6]. Given the livelong course of the disease, lymphedema is a major socio-economic burden [7].

Complex decongestive therapy is still considered the gold standard in the management of lymphedema [8]. This treatment may avoid disease progression, but it cannot offer a cure. In the last decades, reconstructive microsurgical procedures, such as the transfer of vascularized lymph node (VLN) flaps [9], the connection of afferent lymphatic vessels to the venous system (lymphovenous anastomosis) [10], and the transplantation of lymphatic vessels [11] have been introduced. These techniques aim at a functional reconstruction of the lymphatic pathway and, thus, have a curative potential. Recent evidence indicates that the transfer of VLN flaps may be the most powerful approach to re-establish a functional lymphatic drainage [12,13]. These flaps contain a variable number of lymph nodes and perinodal adipose tissue. Since the introduction of VLN flaps in 1979 [14], numerous studies have analyzed the function of transplanted lymph nodes and revealed that they predominantly act as a "lymphatic pump", draining the interstitial lymph through intranodal lymphovenous connections to the venous circulation [13,15]. According to this theory, the efficacy of VLN flaps is positively correlated with their content of lymph nodes [16]. However, this drainage can only take place if the transplanted lymph nodes are functionally integrated into the surrounding host tissue. Importantly, the biological processes underlying functional lymph node integration are complex and not well understood.

Previous preclinical and clinical studies reporting mechanisms of VLN flap integration predominantly focused on the transplanted lymph nodes [13,17]. However, it may be assumed that not only the transplanted lymph nodes but also the perinodal adipose tissue contributes to the restoration of lymphatic function after VLN flap surgery. In the present study, we introduce an experimental model for the assessment of short-term molecular and cellular effects of intraoperative ischemia/reperfusion (IR) on VLN flaps. For this purpose, axillary VLN flaps were dissected in a rat model and exposed to IR. The animal model was validated by means of ultrasound and photoacoustic imaging. Histological, immunohistochemical and Western blot analyses were performed to quantify growth factor expression, microvessel density, cell proliferation, immune cell infiltration, oxidative stress and apoptotic cell death in lymph nodes and the perinodal adipose tissue of VLN flaps.

Results

Animal model

VLN flaps were dissected pedicled on the lateral thoracic vessels in the right axilla of Lewis rats (Fig 1A–1C). After complete dissection, the vascular pedicle of the flaps was clamped for 45 minutes (IR-45) or 120 minutes (IR-120) using microvascular clamps. After releasing the clamps, the skin incisions were closed and the animals were observed for 24 hours. There were no complications with wound healing and all animals showed unrestricted mobilization of the operated limb. Following the IR protocol, VLN flaps were sampled for immunohistochemistry or Western blot (Fig 1D). For the latter, the lymph nodes of individual flaps were microsurgically excised from the perinodal tissue using a stereomicroscope.

Validation of animal model

For an objective in vivo assessment of IR, VLN flaps of the IR-45 and IR-120 groups were analyzed by means of ultrasound and photoacoustic imaging (Fig 2A–2P). During ischemia, sO₂ significantly decreased in the lymph nodes of both groups by ~ 40% when compared to baseline measurements (Fig 2A–2F, 2N and 2P). After reperfusion, sO₂ rapidly returned to baseline levels (Fig 2G–2L, 2N and 2P). There was no statistically significant difference between sO₂ levels of IR-45 and IR-120 lymph nodes (Fig 2N and 2P). In addition, HbT/mm³ was constant throughout the course of the experiments (Fig 2M and 2O). Moreover, the lymph nodes of both groups exhibited comparable HbT/mm³ levels after 24 hours of reperfusion (Fig 2M and 2O). This indicates that the observed changes in tissue oxygenation were not caused by different tissue hemoglobin levels but reduced oxygenated hemoglobin. Finally, stereomicroscopic imaging revealed a dilated perinodal vascular network with small capsular hemorrhages in both ischemia groups (Fig 2Q–2T).

Fig 2 — **A-L** Ultrasound (B-mode) and photoacoustic (HbT, sO₂) imaging of IR-45 and IR-120 axillary VLN flaps before (A and B), during (**C-F**) and after (**G-L**) ischemia (broken line = lymph node). **M-P** Total hemoglobin (HbT/mm³) and oxygen saturation (sO₂ (%)) of IR-45 (M and N, cyan) and IR-120 (O and P, red) lymph nodes at baseline, beginning of ischemia (I0'), after 45 minutes (I45') or 120 minutes (I120') of ischemia as well as directly (R0'), 10 minutes (R10') and 24 hours (R24h) after reperfusion. Mean ± SEM, n = 4, *P < 0.05, n.s. = not significant. **Q-T** Stereomicroscopic images of IR-45 (Q and R) and IR-120 (S and T) VLN flaps. Higher magnification (R and T = inserts of Q and S) reveals a dilated vascular network (arrowheads) with capsular hemorrhages (asterisks). Scale bars: **A-L** = 2 mm, Q and S = 4.5 mm, R and T = 900 μm. IR = ischemia/reperfusion, VLN = vascularized lymph node.

Morphology of VLN flaps

Furthermore, the impact of ischemia on the morphology of axillary VLN flaps was assessed by means of histological and immunohistochemical staining (Fig 3). All groups exhibited a comparable lymph node structure with subcapsular, intermediate and medullary sinuses as well as follicles (Fig 3A–3G), characterized by a LYVE-1⁺ lymphatic network separating these areas (Fig 3B, 3C, 3E and 3G). However, ischemia for 120 minutes was associated with a significant decrease of lymph node cellularity (Fig 3H–3K). In contrast, the perinodal adipose tissue of the IR-45 and IR-120 groups did not exhibit lower cell counts when compared to the control group (Fig 3L).

Fig 3 — **A-G** HE-stained (**A, D** and F) and immunohistochemical (**B, C, E** and G) sections of control (**A-C**), IR-45 (D and E) and IR-120 (F and G) VLN flaps. SS = subcapsular sinus, IS = intermediate sinus, MS = medullary sinus, F = follicle. Arrowhead = LYVE-1⁺ cell-lined sinus. C (insert of B) Asterisk = main efferent lymphatic vessel. **H-J** HE-stained section of follicles of control (H), IR-45 (I) and IR-120 (J) lymph nodes. K Lymph node cellularity (nuclei mm^-2). Mean ± SEM, n = 7–8, *P < 0.05, **P < 0.001. L Adipose tissue cellularity (nuclei mm^-2). Mean ± SEM, n = 7–8, n.s. = not significant. Scale bars: **A, B, D-G** = 300 μm, C = 40 μm, **H-J** = 60 μm. HE = hematoxylin and eosin, LYVE = lymphatic vessel endothelial hyaluronan receptor, VLN = vascularized lymph node.

Microvascular network analysis

VLN flaps were further investigated for alterations of their microvascular network after IR injury. For this purpose, sections were stained with the endothelial cell marker CD31 and the microvessel density of lymph nodes as well as the perinodal adipose tissue was quantified (Fig 4). Lymph nodes of the IR-45 and IR-120 groups exhibited a significantly reduced density of CD31⁺ microvessels when compared to the control group (Fig 4E). In the perinodal adipose tissue, the overall number of microvessels was lower when compared to that within the lymph nodes (Fig 4F). Moreover, the negative IR effect on microvessel density was less pronounced when compared to the changes observed in the nodal component of the flaps (Fig 4F).

Fig 4 — **A-D** Immunohistochemical sections of control (A and B), IR-45 (C) and IR-120 (D) VLN flaps illustrating CD31⁺ microvessels (arrowheads) in lymph nodes. B (insert of A) CD31⁺ paracortical microvessels (arrowheads). Dashed arrows in D = local hemorrhages. E Lymph node microvessel density (mm^-2). Mean ± SEM, n = 7–8, **P < 0.001. **F-H** Immunohistochemical sections of control (F), IR-45 (G) and IR-120 (H) VLN flaps illustrating CD31⁺ microvessels (arrowheads) in the perinodal adipose tissue. I Adipose tissue microvessel density (mm^-2). Mean ± SEM, n = 7–8, *P < 0.05. Scale bars: **A, C** and D = 250 μm, B = 40 μm, **F-H** = 60 μm. VLN = vascularized lymph node.

Expression of lymphangiogenic and angiogenic growth factors

To examine the effects of IR on lymphangiogenesis and angiogenesis, the marker proteins vascular endothelial growth factor (VEGF)-C, VEGF-D and VEGF-A were analyzed by means of Western blot (Fig 5A–5D). For protein expression analyses, lymph nodes and perinodal adipose tissue of individual flaps were sampled separately. We found a significantly increased expression of the lymphangiogenic growth factor VEGF-D in lymph nodes (Fig 5C) as well as perinodal adipose tissue (Fig 5D) after 120 minutes ischemia when compared to samples of the control and IR-45 group. Surprisingly, VEGF-C expression was not upregulated after 120 minutes ischemia. Furthermore, the expression of the angiogenic growth factor VEGF-A was significantly higher in the lymph nodes of the IR-120 group but not in the corresponding perinodal adipose tissue (Fig 5C and 5D). The original Western blots are provided in the supplementary information.

Fig 5 — **A-D** Western blots (A and B) with quantification (C and D) of VEGF-C, VEGF-D and VEGF-A expression of control (white), IR-45 (cyan) and IR-120 (red) VLN flaps. Bands were cropped from different parts of the same gel. Panels A and C represent analyses of lymph nodes and panels B and D analyses of adipose tissue. Mean ± SEM, n = 4, *P < 0.05, **P < 0.001, n.s. = not significant. **E-J** Western blots (E and F) with quantification of iNOS (G and I) and p-eNOS/eNOS (H and J) expression of control (white), IR-45 (cyan) and IR-120 (red) VLN flaps. Bands were cropped from different parts of the same gel. Panels **E, G** and H represent analyses of lymph nodes and panels **F, I** and J analyses of adipose tissue. Mean ± SEM, n = 4, *P < 0.05, n.s. = not significant. IR = ischemia/reperfusion, iNOS = inducible nitric oxide synthase (NOS), eNOS = endothelial NOS, p-eNOS = phospho-eNOS, VEGF = vascular endothelial growth factor, VLN = vascularized lymph node.

Expression of iNOS, eNOS and p-eNOS

Additional Western blot analyses were performed to analyze the expression of the inducible nitric oxide (NO)-synthase (iNOS) and the constitutively expressed endothelial NO-synthase (eNOS) and its phosphorylated form phospho-endothelial NO-synthase (p-eNOS). We found that neither iNOS nor p-eNOS/eNOS expression were significantly upregulated in ischemic lymph nodes when compared to lymph nodes of the control group (Fig 5E, 5G and 5H). In contrast, perinodal adipose tissue undergoing 120 minutes ischemia exhibited a significantly increased p-eNOS/eNOS ratio when compared to adipose tissue of the control and IR-45 group, indicating adipose tissue susceptibility to IR injury (Fig 5F, 5I and 5J). The original Western blots are provided in the supplementary information.

Oxidative stress, cell proliferation, cell apoptosis

To quantify oxidative stress, cellular proliferation and apoptotic cells in ischemic VLN flaps sections were stained with antibodies against the heat-shock protein heme oxygenase-1 (HO-1), proliferating cell nuclear antigen (PCNA) as well as cleaved caspase-3 (Casp-3). Immunohistochemical analyses revealed a significantly higher fraction of cells expressing HO-1 in lymph nodes of the IR-120 group when compared to control and IR-45 lymph nodes (Fig 6A–6D). Moreover, lymph nodes contained a markedly higher number of PCNA⁺ cells (Fig 6E–6H) and Casp-3⁺ cells (Fig 6I–6L) after 120 minutes of ischemia. The majority of the proliferating and apoptotic cells were lymphocytes, as verified by microscopic assessment of cell morphology. Surprisingly, immunohistochemical investigations of the perinodal adipose tissue did not reveal significantly increased oxidative stress, cell proliferation as well as apoptotic cell death after 45 and 120 minutes of ischemia, as indicated by HO-1, PCNA and Casp-3 stainings (Fig 7A–7L).

Fig 6 — **A-D** Immunohistochemical detection of HO-1⁺ cells (arrowheads) in control (A), IR-45 (B) and IR-120 (C) lymph nodes. D Quantitative analysis of HO-1⁺ cells (%). **E-H** Immunohistochemical detection of PCNA⁺ cells (arrowheads) in control (E), IR-45 (F) and IR-120 (G) lymph nodes. H Quantitative analysis of PCNA⁺ cells (%). **I-L** Immunohistochemical detection of Casp-3⁺ cells (arrowheads) in control (I), IR-45 (J) and IR-120 (K) lymph nodes. L Quantitative analysis of Casp-3⁺ cells (%). **M-P** Immunohistochemical detection of CD68⁺ macrophages (arrowheads) in control (M), IR-45 (N) and IR-120 (O) lymph nodes. P Quantitative analysis of CD68⁺ cells (mm^-2). **Q-T** Immunohistochemical detection of MPO⁺ neutrophilic granulocytes (arrowheads) in control (Q), IR-45 (R) and IR-120 (S) lymph nodes. T Quantitative analysis of MPO⁺ cells (mm^-2). Mean ± SEM, n = 6–8, *P < 0.05, **P < 0.001. Scale bars: **A-C**, **E-G** and **I-K** = 25 μm, **M-O** and **Q-S** = 50 μm. Casp-3 = cleaved caspase-3, HO = heme oxygenase, IR = ischemia/reperfusion, MPO = myeloperoxidase, PCNA = proliferating cell nuclear antigen, VLN = vascularized lymph node.

Fig 7 — **A-D** Immunohistochemical detection of HO-1⁺ cells (arrowheads) in perinodal adipose tissue of control (A), IR-45 (B) and IR-120 (C) VLN flaps. D Quantitative analysis of HO-1⁺ cells (%). **E-H** Immunohistochemical detection of PCNA⁺ cells (arrowheads) in perinodal adipose tissue of control (E), IR-45 (F) and IR-120 (G) VLN flaps. H Quantitative analysis of PCNA⁺ cells (%).I-L Immunohistochemical detection of Casp-3⁺ cells (arrowheads) in perinodal adipose tissue of control (I), IR-45 (J) and IR-120 (K) VLN flaps. L Quantitative analysis of Casp-3⁺ cells (%). **M-P** Immunohistochemical detection of CD68⁺ macrophages (arrowheads) in perinodal adipose tissue of control (M), IR-45 (N) and IR-120 (O) VLN flaps. P Quantitative analysis of CD68⁺ cells (mm^-2). **Q-T** Immunohistochemical detection of MPO⁺ neutrophilic granulocytes (arrowheads) in perinodal adipose tissue of control (Q), IR-45 (R) and IR-120 (S) VLN flaps. T Quantitative analysis of MPO⁺ cells (mm^-2). Mean ± SEM, n = 7–8, n.s. = not significant. Scale bars = 50 μm. Casp-3 = cleaved caspase-3, HO = heme oxygenase, IR = ischemia/reperfusion, MPO = myeloperoxidase, PCNA = proliferating cell nuclear antigen, VLN = vascularized lymph node.

Immune cell infiltration

Finally, for the quantification of immune cell infiltration of VLN flaps sections were stained with CD68 and myeloperoxidase (MPO) for the detection of macrophages and neutrophilic granulocytes, respectively. Prolonged ischemia of VLN flaps was associated with an increased immune cell infiltration in the nodal flap component. Accordingly, lymph nodes of the IR-120 group exhibited a significantly higher number of CD68⁺ macrophages (Fig 6M–6P) and MPO⁺ neutrophilic granulocytes (Fig 6Q–6T) when compared to control lymph nodes. In contrast, analyses of the perinodal adipose tissue did not show significantly higher numbers of infiltrating macrophages and neutrophilic granulocytes in the IR-45 and IR-120 groups when compared to the non-ischemic control (Fig 7M–7T).

Discussion

Cancer-related chronic lymphedema is a highly prevalent disease with an underestimated socio-economic burden. In the last decades, promising microsurgical treatment strategies have been introduced with the aim to restore physiological lymphatic drainage. Among these, VLN flaps exhibit a particularly high potential to tackle the pathophysiological changes of chronic lymphedema. Despite recent hallmark studies describing the mechanism how VLN flaps ameliorate lymphatic drainage [13,15], the biological processes underlying flap integration remain a subject of debate.

In the present investigation, we analyzed the short-term effect of IR injury on VLN flaps. To prevent challenging repetitive monitoring of rodent anastomoses, we used an IR model with temporary clamping of the vascular pedicle. In clinical practice, intraoperative ischemia of flaps commonly ranges between 45 and 120 minutes. Consequently, we applied ischemia for 45 and 120 minutes to generate data of translational interest. Photoacoustic imaging revealed a consistent reduction of oxygen saturation in VLN flaps after 45 and 120 minutes ischemia, indicating a high reliability of the model. We did not find a compensatory hyper-oxygenation at the onset of reperfusion. Furthermore, stereomicroscopic assessment of VLN flaps after 45 and 120 minutes ischemia did not reveal different alterations of the superficial vascular network. However, detailed histological and immunohistochemical analyses showed that 120 minutes ischemia may already be critical for the physiological lymph node architecture [18]. In fact, we found a significantly lower lymph node cellularity after 120 minutes ischemia when compared to the control and IR-45 samples, leading to a ~ 50% reduction of cell nuclei in lymph nodes undergoing prolonged ischemia. In contrast, the perinodal adipose tissue of VLN flaps undergoing IR injury was not characterized by lower cell counts when compared to control samples, indicating a higher susceptibility of the nodal component to ischemic damage.

Functional lymph node integration of VLN flaps critically depends on the expression of lymphangiogenic growth factors, such as VEGF-C and VEGF-D [19]. For instance, exogenous VEGF-C enhances lymphatic vessel formation and function and is crucial to preserve lymph node histology of VLN in pigs [20]. Transferred lymph nodes have been identified as an endogenous source for lymphangiogenic growth factor expression [17]. Surprisingly, little effort has been directed towards the clarification of the function of the adipose tissue within VLN flaps. The perinodal adipose tissue contains a dense lymphatic network and is rich of stem cells and inflammatory cells, which may support lymph node integration after VLN flap transfer. According to this assumption, Aschen et al. reported high VEGF-C expression in adipose tissue of the recipient site after avascular lymph node transplantation in mice [21]. Based on these findings, we speculated that the perinodal adipose tissue of VLN flaps may be equally or even more important for intraoperative endogenous growth factor expression compared with the nodal component.

To test this hypothesis, Western blot analyses were performed to evaluate the immediate effect of IR on growth factor expression in VLN flaps. We found a markedly increased expression of VEGF-D in lymph nodes and perinodal adipose tissue of VLN flaps after 120 minutes ischemia, whereas VEGF-C expression was not upregulated in this group. Furthermore, we observed an upregulated VEGF-A expression in lymph nodes after 120 minutes ischemia and a high VEGF-A expression in the perinodal adipose tissue of all groups. These findings are important because VEGF-D and VEGF-A overexpression can lead to blood and lymphatic vessel hyperpermeability with seroma formation through VEGF receptor-2 (VEGFR-2) signaling [22]. In line with our findings, previous clinical studies also revealed increased VEGF-D levels in axillary seroma fluid after VLN flap surgery [23,24]. However, these studies found even higher levels of VEGF-C, which is commonly accepted as the most important pro-lymphangiogenic growth factor concerning reconstructive lymphatic surgery [22]. Importantly, analyzing postoperative seroma fluid does not allow identifying the source of growth factor expression. Hence, even though limited by a small sample size, our analysis contributes to the understanding of perioperative lymphangiogenic growth factor expression during VLN flap surgery.

Noteworthy, the increased VEGF-A expression in IR-exposed lymph nodes was not associated with a higher microvessel density, as indicated by additional immunohistochemical analyses. In fact, lymph nodes undergoing 45 and 120 minutes of ischemia even contained less CD31⁺ microvessels when compared to non-ischemic controls. From a biological point of view, this finding is not surprising because IR-related tissue damage leads to the loss of microvascular structures and local hypoxia. Consequently, angiogenic growth factors such as VEGF-A are upregulated, promoting angiogenic neovessel growth. However, angiogenesis is a time-consuming process with a slow growth rate of new microvessels of ~ 5 μm per hour [25]. Hence, 24 hours of reperfusion may be too short to demonstrate an increased vascularization. In contrast, the loss of microvessels in the perinodal adipose tissue was less pronounced, providing further evidence that this flap component is less susceptible to IR-induced tissue injury.

Of interest, we detected an activation of eNOS in IR-exposed perinodal adipose tissue. This result supports the view of Gust et al., who recently suggested an active role of adipose tissue in driving the inflammatory response after IR injury [26]. They showed an overexpression of stress and inflammatory markers as well as inflammatory cell infiltration of mature adipose tissue following IR injury in a mouse model. Other authors found that severe ischemia is associated with loss of mature adipocytes, which are replaced with new adipocytes derived from resident adipose-derived progenitor cells [27]. These progenitor cells contribute to adipogenesis, angiogenesis and, presumably, also lymphangiogenesis. Despite moderate changes in cellularity and microvessel density observed in the present investigation, it may be assumed that the perinodal adipose tissue of VLN flaps exerts angiogenic and lymphangiogenic effects during the initial days to weeks after transplantation, contributing to the formation of a microvascular and lymphatic capillary network of VLN flaps. Remarkably, subsequent immunohistochemical analyses of the perinodal adipose tissue did not reveal a higher percentage of HO-1⁺, PCNA⁺ or Casp-3⁺ cells. Furthermore, the number of infiltrating inflammatory cells was not markedly higher after 45 or 120 minutes of ischemia compared with the control group. This may be explained with technical differences of the experimental models. For instance, Gust et al. used an IR model with magnetic skin compression [26]. In contrast, the herein used IR model exposes the adipose tissue to indirect ischemia without mechanical manipulation. Hence, the less traumatic induction of ischemia might also result in fewer IR-associated tissue alterations.

Finally, immunohistochemical analyses revealed that lymph nodes of VLN flaps are prone to IR injury, as indicated by a higher percentage of HO-1⁺ cells after 120 minutes ischemia. Moreover, lymph nodes undergoing 120 minutes ischemia also contained markedly higher numbers of proliferating PCNA⁺ and apoptotic Casp-3⁺ cells as well as CD68⁺ macrophages and MPO⁺ neutrophilic granulocytes when compared to lymph nodes of the IR-45 and control group. In contrast to our findings, the current opinion on critical ischemia time of VLN flaps is four hours as evaluated for inguinal VLN flaps in a rat model [28,29]. However, a recent analysis of inflammatory biomarkers revealed that significant IR injury can occur with as little as two hours of ischemia [30]. In line with this, the histological and immunohistochemical findings of the present study support the assumption that lymph nodes may be more susceptible to IR injury as previously assumed and already undergo significant histological changes after two hours of ischemia.

This study has important limitations. First, we only analyzed short-term cellular and molecular effects of IR injury on VLN flaps. The herein used animal model is not suitable to investigate the long-term effect of upregulated growth factors or cell damage on the integration and function of VLN flaps. This research question must be addressed in future experiments. Second, we only analyzed a small selection of growth factors involved in VLN flap function. Nevertheless, our findings contribute to the understanding of the initial phase of IR injury in lymph nodes and perinodal adipose tissue. Finally, in clinical practice, VLN flaps are performed with microsurgical anastomoses. In the present study, we used in situ clamping of the vascular pedicle to induce IR injury. However, in the experimental setting and particularly when working with rodent models, this technique may be more reliable compared with microsurgical VLN flap transfer because it eliminates the potential bias of microvascular complications.

In summary, the present study demonstrates that VLN flaps are remarkably prone to IR injury. In particular, prolonged intraoperative ischemia of 120 minutes is already associated with a significant reduction of cellularity and vascularization of lymph nodes. Moreover, we found an increased expression of the growth factors VEGF-D and VEGF-A as well as apoptotic cell death and immune cell infiltration of the nodal flap component. In contrast, the perinodal adipose tissue is less susceptible to IR injury but contributes to lymphangiogenic and angiogenic growth factor expression immediately after VLN flap surgery. From a translational point of view, further investigations are required to elucidate the long-term effect of IR injury on the functional capacity of VLN flaps.

Material and methods

Animals

The experiments were performed with 44 Lewis rats (Janvier Labs, Le Genest-Saint-Isle, France) exhibiting an age of 17 ± 1 weeks and a body weight of 397 ± 8 g. The animals were housed one per cage under a 12-hour day/night cycle and were fed ad libitum with water and standard pellet food (Altromin, Lage, Germany). All experiments were conducted in accordance with the European legislation on the protection of animals (Directive 2010/63/EU) and the National Institutes of Health guidelines on the care and use of laboratory animals (National Institutes of Health publication #85–23 Rev. 1985). They were approved by the local governmental animal protection committee (Landesamt für Verbraucherschutz, Saarbrücken, Germany; Permission number: 67/2015).

Surgery

Flap dissection was performed by modifying a recently published model [31]. The rats were anesthetized by intraperitoneal injection of ketamine (80 mg/kg body weight; Ursotamin; Serumwerk Bernburg AG, Bernburg, Germany) and xylazine (6 mg/kg body weight; Rompun; Bayer, Leverkusen, Germany). After depilation, the animals were placed in supine position on a heated operation stage. After a skin incision in the axilla (Fig 1A), the pectoralis major muscle was retracted and the axillary neurovascular bundle was identified. Following identification of the lateral thoracic vessels (Fig 1B), the adjacent lymph node flap was circumferentially dissected with destruction of all afferent and efferent lymphatic vessels. Care was taken to preserve the brachial plexus. After complete dissection of the flap (Fig 1C), the lateral thoracic artery and vein (i.e. the flap pedicle) were clamped in situ using microvascular clamps (S&T AG Microsurgical Instruments, Neuhausen, Switzerland) according to the experimental protocol. After releasing the clamps and verification of the establishment of blood flow, the incision was closed with 5/0 monofilament (Prolene; Ethicon, Johnson & Johnson Medical GmbH, Norderstedt, Germany). Postoperative analgesia was provided by subcutaneous buprenorphine (0.05 mg/kg body weight; Buprenovet; Bayer Vital GmbH, Leverkusen, Germany) and tramadol hydrochloride (40 mg per 100 mL drinking water; Tramal; Grünenthal GmbH, Aachen, Germany). Wounds and animal behaviour were regularly checked until the rats were killed 24 hours after surgery.

Ultrasound, photoacoustic imaging and stereomicroscopy

Rats with a dissected axillary VLN flap were anesthetized with 1.5% isoflurane and put on a heated stage. After covering the flap with ultrasound coupling gel (Aquasonic 100; Parker, Fairfield, NJ, USA), photoacoustic imaging was performed by means of a Vevo LAZR system (FUJIFILM Visualsonics Inc., Toronto, ON, Canada) and a real-time microvisualization LZ550 linear-array transducer (FUJIFILM Visualsonics Inc.) with a center frequency of 40 MHz. Heart and breathing rate were constantly monitored and the body temperature was maintained at 36°C (THM100; Indus Instruments, Houston, TX, USA). For three-dimensional imaging, the ultrasound probe was driven over the entire flap by a linear motor to acquire two-dimensional images at parallel and uniformly spaced, 150 μm-sized intervals. The two-dimensional image planes were then stitched together enabling rapid three-dimensional image reconstruction, displaying a dynamic cube view format, as described previously [32]. In addition, Oxy-Hemo-mode photoacoustic images were taken at two wavelengths (i.e. 750 nm and 850 nm) with a two-dimensional photoacoustic gain of 44 dB and a hemoglobin threshold of 20 dB. To measure the total hemoglobin signal [HbT/mm³] within lymph nodes, all detected signals at the two wavelengths where divided by the volume of the analyzed lymph nodes. Furthermore, the oxygen saturation [sO₂] of individual lymph nodes was evaluated in %, as described previously [33,34]. All data values were analyzed using the Vevo LAB 1.7.2 software (FUJIFILM Visualsonics Inc.).

For stereomicroscopy, excised VLN flaps were placed under a stereomicroscope (Leica M651; Leica, Wetzlar, Germany) and the recorded images were transferred to a DVD system.

Western blot

After shock freezing in liquid nitrogen and storage at—80°C, the lymph node and adipose tissue samples were lysed by homogenization using a lysis buffer to extract the whole cell protein fraction with additional protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1:75 v/v Protease Inhibitor Cocktail, 1:100 v/v Phosphatase Inhibitor Cocktail 2; Sigma-Aldrich, Taufkirchen, Germany). Lysates were then collected and centrifuged at 4°C and 13 000 xg for 30 minutes. Supernatants were saved as whole protein extracts and protein concentrations were analyzed photometrically using the Lowry method. Then, 30 μg protein per lane were separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, München, Germany). Subsequently, membranes were incubated with the following primary antibodies over night at 4°C, followed by a supplemental incubation for 3 hours at room temperature: VEGF-C (1:300; Abcam, Cambridge, UK), VEGF-D (1:300; Abcam), VEGF-A (1:100; Santa Cruz Technology, Heidelberg, Germany), iNOS (1:300; Abcam), eNOS (1:300; BD Biosciences, Heidelberg, Germany) and p-eNOS (1:500; Cell Signaling Technology, Frankfurt, Germany). Corresponding horseradish peroxidase-conjugated secondary antibodies (1:5 000; GE Healthcare, Freiburg, Germany or 1:1 000; R&D Systems, Wiesbaden, Germany) were then attached at room temperature for 1.5 hours. Protein expression was visualized with enhanced chemiluminescence (ECL Western Blotting Analysis System, GE Healthcare) and analyzed with an ECL ChemoCam Imager (Chemostar and LabImage 1D software; Intas Science Imaging Instruments, Göttingen, Germany).

Histology and immunohistochemistry

After excision and stereomicroscopy, flaps were fixed in 4% formalin, embedded in paraffin, and cut into 3-μm thick sections. Individual sections were stained with hematoxylin and eosin (HE) according to standard procedures. Using a BX60 microscope (Olympus, Hamburg, Germany) and the imaging software cellSens Dimension 1.11 (Olympus), the cellularity (number of nuclei, given in mm^-2) of individual lymph nodes was assessed at 800x magnification in 5 randomized regions of interest (ROIs) per section and of the perinodal adipose tissue at 200x magnification in 4 randomized ROIs per section.

For the immunohistochemical detection of lymphatic vessels, additional sections were stained with a polyclonal rabbit antibody against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) (1:600; Abcam). A goat anti-rabbit IgG Alexa555 antibody (1:200; Life Technologies, Eugene, OR, USA) served as secondary antibody. Cell nuclei were stained with Hoechst 33342 (2 μg/mL; Sigma-Aldrich) for image merging.

For the immunohistochemical detection of microvessels, sections were stained with a monoclonal rat antibody against the endothelial cell marker CD31 (1:100; Dianova, Hamburg, Germany). A goat anti-rat IgG Alexa555 antibody (1:200; Thermo Fisher Scientific GmbH, Dreieich, Germany) was used as secondary antibody. Cell nuclei were stained with Hoechst 33342 (2 μg/mL; Sigma-Aldrich) for image merging. The density of CD31⁺ microvessels (given in mm⁻²) of individual lymph nodes was assessed at 400x magnification within 5 randomized ROIs and of the perinodal adipose tissue at 200x magnification within 4 randomized ROIs per section.

Further sections were stained with a polyclonal rabbit antibody against HO-1 (1:100; Enzo Life Sciences GmbH, Lörrach, Germany). A peroxidase-labeled goat anti-rabbit IgG antibody (1:200; Dianova) served as secondary antibody. Additional sections were stained with a monoclonal mouse antibody PCNA (1:100; Agilent, Hamburg, Germany). A peroxidase-labeled goat anti-mouse IgG antibody (1:200; Dianova) served as secondary antibody. Finally, sections were stained with a polyclonal rabbit antibody against Casp-3 (1:100; New England BioLabs GmbH, Frankfurt, Germany) followed by a biotinylated goat anti-rabbit IgG antibody (ready-to-use; Abcam). The biotinylated antibodies were detected by peroxidase-labeled streptavidin (ready-to-use; Abcam). 3-Amino-9-ethylcarbazole (Abcam) was used as chromogen for PCNA-stained sections and 3,3-diaminobenzidine (Sigma-Aldrich) was used as chromogen for Casp-3 and HO-1-stained sections. The number of HO-1⁺, PCNA⁺ and Casp-3⁺ cells (given in % of all cells) of individual lymph nodes was analyzed at 400x magnification in 6 randomized ROIs per section and of the perinodal adipose tissue at 200x magnification in 4 randomized ROIs per section.

Finally, sections were stained with a polyclonal rabbit antibody against CD68 (1:50; Abcam) and a polyclonal rabbit antibody against MPO (1:100, Abcam). This was followed by a biotinylated goat anti-rabbit IgG antibody (ready-to-use; Abcam), which was detected by peroxidase-labeled streptavidin (ready-to-use; Abcam). 3-Amino-9-ethylcarbazole (Abcam) was used as chromogen. Cell infiltration (given in mm^-2) of individual lymph nodes was quantified at 400x magnification in 5 randomized ROIs per section and of the perinodal adipose tissue at 200x magnification in 4 randomized ROIs per section. All quantitative analyses were performed with the software package ImageJ.

Experimental protocol

For photoacoustic validation of the animal model, axillary VLN flaps of 8 rats were scanned before ischemia, at the beginning of ischemia, after 45 minutes (n = 4) or 120 minutes (n = 4) of ischemia as well as directly, 10 minutes and 24 hours after reperfusion. The animals were sacrificed after the last scan.

For histology and immunohistochemistry, VLN flaps were dissected in 24 rats. In the control group (n = 8), no ischemia was applied and the wounds were directly closed after complete flap dissection. In the IR-45 (n = 8) and IR-120 group (n = 8), the vascular pedicle was clamped for 45 and 120 minutes, respectively. The animals were sacrificed 24 hours after skin suture (control) or onset of reperfusion (IR-45/IR-120) and tissue specimens were processed for histological and immunohistochemical analyses.

For Western blotting, VLN flaps were dissected in 12 rats. Following the IR protocol for the control (n = 4), IR-45 (n = 4) and IR-120 (n = 4) group, the animals were sacrificed and the lymph nodes and the perinodal adipose tissue were excised and processed for protein expression analyses.

Statistics

Data were analyzed for normal distribution and equal variance. Parametric data were compared using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. In case of non-parametric distribution, groups were analyzed with the Kruskal-Wallis test followed by Dunn’s post hoc test. To test for time effects within groups, ANOVA for repeated measurements was applied followed by Tukey’s post hoc test. Data are given as scatter dot plots with mean ± standard error of the mean (SEM). Statistical significance was accepted for P < 0.05. The statistical analysis was performed using Prism 7.0d (GraphPad Software, Inc.).

Supporting information

S1 Raw images

(PDF)

Click here for additional data file.^{(1.3MB, pdf)}

Acknowledgments

We are grateful for the excellent technical assistance of Janine Becker, Caroline Bickelmann, Ruth Nickels and Julia Parakenings.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

YES 1. FSF was supported by Deutsche Gesellschaft für Lymphologie (DGL) 2. Grant number: Fo0315 3. https://www.dglymph.de/aktuelles 4. No, sponsor not involved in research.

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PLoS One. doi: 10.1371/journal.pone.0239517.r001

Decision Letter 0

Ferenc Gallyas, Jr

27 Sep 2019

PONE-D-19-21979

Molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

PLOS ONE

Dear Dr. Frueh,

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: The authors evaluated the molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flap in a rat model. After ischemia treatment of 45 min or 120 min and followed by reperfusion for 24 hours, the oxygen content, level of oxidative stress and growth factors were examined. The authors separate lymph nodes and perinodal adipose tissue to observe the ischemia/reperfusion effects. The number of proliferating and apoptotic cells were increased after ischemia/reperfusion treatment. Some data still required to be added to make the manuscript more complete.

Specific comments:

a. In Fig 3, the authors observed the expression of LYVE. Did any changes in the expression of LYVE under different ischemia time or in the location of LYVE expression? Any scientific relevant of using LYVE in this staining, since the authors only observed the structure of the lymph node under ischemia treatment.

b. The results on Fig. 4 presented the increase level of VEGF-A. Did the authors stain for CD31 or other angiogenesis markers to evaluate whether the blood vessel density were also increased in lymph nodes or adipose tissue?

c. The increase number of proliferating cells were identified in Fig. 5 by using PCNA+ marker. However, the other results indicated the cellularity were decreased after 45 min/120 min ischemia and 24 hours reperfusion treatment (in Fig. 3). What kind of cells were the proliferating cells? And what kind of cell were the caspas 3 labeled?

d. How the authors choose the 45 and 120 min as the ischemia time and 24 hours as the reperfusion time?

e. In the Discussion, the authors emphasized on the different effects after ischemia treatment on lymph nodes and adipose tissue. However, the ischemia effect on lymph nodes were presented in the Fig. 4. There were no cell cellularity, HO-1+, PCNA+ or immune cell examination on adipose tissue.

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PLoS One. 2020 Oct 6;15(10):e0239517. doi: 10.1371/journal.pone.0239517.r002

Author response to Decision Letter 0

26 Jan 2020

Review of the manuscript PONE-D-19-21979

Reply to the comments of reviewer 1

We thank the reviewer for the fair and constructive comments. Please find our point-by-point reply in the following.

Specific comments:

Reviewer comment 1

In Fig 3, the authors observed the expression of LYVE. Did any changes in the expression of LYVE under different ischemia time or in the location of LYVE expression? Any scientific relevant of using LYVE in this staining, since the authors only observed the structure of the lymph node under ischemia treatment.

We thank the reviewer for this important comment. The reviewer is correct that LYVE-1 staining in this part of our investigation was only used for descriptive purposes and not for a quantitative analysis of lymphatic vessel density. In this context it should be noted that the expression of LYVE-1 in the lymphatic sinuses of lymph nodes is not limited to lymphatic endothelial cells but is also observed in other cell types, such as reticular cells (Bai et al., 2011). Consequently, a quantification of the lymphatic vascular network (ie, the sinuses) within individual lymph nodes is not appropriate using LYVE-1 as marker.

Reference

Bai Y, Wu B, Terada N, Ohno N, Saitoh S, Saitoh Y, Ohno S. Histological study and LYVE-1 immunolocalization of mouse mesenteric lymph nodes with "in vivo cryotechnique". Acta Histochem Cytochem. 2011;44:81-90.

Reviewer comment 2

The results on Fig. 4 presented the increase level of VEGF-A. Did the authors stain for CD31 or other angiogenesis markers to evaluate whether the blood vessel density were also increased in lymph nodes or adipose tissue?

According to the comment of the reviewer, we have performed additional immunohistochemical stainings of VLN flaps using the endothelial cell marker CD31 to quantify the microvessel density (mm-2) within individual lymph nodes and the perinodal adipose tissue. Interestingly, lymph nodes undergoing 45 and 120 minutes of ischemia exhibited a significantly reduced microvessel density when compared to non-ischemic controls. In contrast, the microvascular network of the perinodal adipose tissue was less sensitive to ischemia/reperfusion injury. This novel information is now included in the revised version of our manuscript (see Figure 4; Results, page 6/7, lines 171-188 as well as Discussion page 12/13, lines 341-347; marked in yellow)

Reviewer comment 3

The increase number of proliferating cells were identified in Fig. 5 by using PCNA+ marker. However, the other results indicated the cellularity were decreased after 45 min/120 min ischemia and 24 hours reperfusion treatment (in Fig. 3). What kind of cells were the proliferating cells? And what kind of cell were the caspas 3 labeled?

The reviewer is correct that despite an overall reduction of cellularity, we observed an increasing number of proliferating cells in the lymph nodes of ischemic VLN flaps. However, this is a common finding after ischemia/reperfusion injury. Due to technical issues, we were not able to further characterize PCNA+ and caspase-3+ cells with co-stainings using lymphocyte-specific antibodies. Instead, we have performed a morphological analysis of individual PCNA+ and caspase-3+ cells within ischemic lymph nodes and found that the majority of them were lymphocytes. This is in line with the findings of a recently published study by Perrault et al. (2020), who reported a particularly high sensitivity of the immune cell component of lymph nodes to ischemia reperfusion injury. This novel information is now included in the revised version of our manuscript (see Results, page 9, lines 234-236; marked in yellow).

Reference

Perrault DP, Lee GK, Bouz A, Sung C, Yu R, Pourmoussa AJ, Park SY, Kim GH, Jiao W, Patel KM, Hong YK, Wong AK. Ischemia and reperfusion injury in superficial inferior epigastric artery-based vascularized lymph node flaps. PLoS One. 2020;15:e0227599.

Reviewer comment 4

How the authors choose the 45 and 120 min as the ischemia time and 24 hours as the reperfusion time?

We chose the ischemia time based on realistic clinical scenarios. In fact, 45 minutes represent an uncomplicated microvascular VLN flap transplantation and 120 minutes represent a difficult or suboptimal microvascular procedure. The reperfusion time was chosen based on our previous experience with ischemia/reperfusion experiments and it has recently been shown that shorter reperfusion times may reduce the biological reaction to ischemia of VLN flaps (Perrault et al., 2020).

Reference

Reviewer comment 5

In the Discussion, the authors emphasized on the different effects after ischemia treatment on lymph nodes and adipose tissue. However, the ischemia effect on lymph nodes were presented in the Fig. 4. There were no cell cellularity, HO-1+, PCNA+ or immune cell examination on adipose tissue.

According to the comment of the reviewer, we have performed an additional immunohistochemical analysis of the perinodal adipose tissue, evaluating the fraction of HO-1+, PCNA+ and caspase-3+ cells as well as the number of infiltrating CD68+ macrophages and MPO+ neutrophilic granulocytes. Of interest, we found that the cells within the perinodal adipose tissue were not markedly affected by 45 or 120 minutes of ischemia. This indicates again a higher resistance of adipose tissue to ischemia/reperfusion injury when compared to the lymph node component of VLN flaps. This novel information is now included in the revised version of our manuscript (Figure 7; see Results, page 9, lines 236-239 and lines 247-250 as well as page 10, lines 267-281; see Discussion, page 13/14, lines 369-374; marked in yellow).

Two new co-authors from the Division of Plastic Surgery and Hand Surgery, University Hospital Zurich and from the Institute for Clinical & Experimental Surgery, Saarland University, markedly contributed to our novel immunohistochemical analyses. Accordingly, both co-workers have been included in the author list of the revised manuscript (see page 1, lines 6 and 7; see page 23, lines 632 and 636; marked in yellow).

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(31.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0239517.r003

Decision Letter 1

Jianhong Zhou

7 May 2020

PONE-D-19-21979R1

Molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

PLOS ONE

Dear Dr. Frueh,

We would appreciate receiving your revised manuscript by Jun 20 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.

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Kind regards,

Jianhong Zhou

Associate Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors evaluated the molecular and cellular effects of ischemia on vascularized lymph node flap. After ischemia of 45 min or 120 min and followed by reperfusion for 24 hours., the oxygen content, the level of oxidative stress and growth factors were examined. The authors separate lymph nodes and perinodal adipose tissue to observe the ischemia effect. The number of proliferating and apoptotic cells were increased after ischemia/reperfusion treatment. The authors made much revisions regarding the reviewers’ comments. Some questions remain as follows:

1. In the result section (page 5), “r and t = 900 μm“, and in the figure legend of Fig. 2, “R and T= 900 μm” are not compatible.

2. On page 6, “ In the IR-120 group, however, we observed a partial loss of the lymph node architecture (Figs. 3F and 3G)”, which particular part was lost?

3. In Fig. 4D, the CD 31 staining seems more abundant by compared to the other two groups, which was inconsistent to the results of Fig. 4 E.

4. According to the discussion, authors referenced Gust’s paper indicated “an active role of adipose tissue in driving the inflammatory response after IR injury [26]. The overexpression of stress and inflammatory markers as well as inflammatory cell infiltration of mature adipose tissue following IR injury. But other authors found that severe ischemia is associated with loss of mature adipocytes, which are replaced with new adipocytes derived from resident adipose-derived progenitor cells [27]. “

Since the results showing a higher level of eNOS presented in the perinodal adipose tiåssue in Fig. 6, there was no significance cell proliferation, apoptosis, no oxidative stress in Fig. 7, which were inconsistent with the results from the reference that the authors provided. How to explain these findings?

5. The results indicated an increasing level of VEGF-A after ischemia condition (Fig. 5). The results were not consistent with that in Fig. 4, in which the micro-vessel density was decreased after ischemia condition.

6. The staining of CD31 in Fig. 4D was not compatible with Figs. 4E and 4F.

Reviewer #2: This manuscript describes the results of a preclinical study analyzing the immediate molecular and cellular effects after ischemia-reperfusion injury on vascularized lymph node transfers. It analyzes various lymphangiogenic growth factors, as well as it evaluates the morphology, proliferation, apoptosis, and infiltration of immune cells in the lymph nodes and perinodal adipose tissue. For this study the authors have used a rat model of vascularized lymph node flap pedicled on the lateral thoracic vessels.

Overall, this is a good research set-up which is well described as well as conducted. However, there are some major and minor revisions to be done.

1. Indicate in the title, in the abstract and when the objective of the study is stated at the end of the introduction, that the main goal is to investigate the “immediate” or “initial” changes at the cellular and molecular level after performing lymph node transfers, since the study ends at 24h postop. Findings may be interpreted as biomarkers to identify the initial phase of I/R injury in lymph nodes and perinodal tissue.

2. The abstract does not indicate which animal model or which lymph nodes are studied, nor the number of animals, nor their distribution in the study groups. Please modify it accordingly.

3. Please modify the image Figure 1.B as it is not clearly seen.

4. In the discussion please focus more on your actual results and their interpretation. This section partially sounds like an introduction. You may move parts to the introduction if needed.

5. In the final paragraph of the discussion (conclusion) try to be more specific and summarize the significant results you have obtained in this study. This will help other researchers and readers to get the most relevant results of this study.

6. It is important that readers keep in mind the limitations of this study. Include a summary of the main ones and explain them in the discussion. Examples:

- There are numerous growth factors that were not included in the analysis, in this sense, we should not exclude the importance of other biomarkers.

- Vascularized lymph node transfers are performed using microsurgical anastomoses, however in this study, VLNTs have been simulated by clamping the vessels.

- The current literature indicates that the critical ischemia time in lymph node transfers is 4h, however in this study a shorter ischemic insult has been studied.

7. In the Material and Methods section, it is indicated that an injectable anesthetic protocol (Ketamine and xylazine) is used during surgery, but during the ultrasound, photoacoustic imaging and stereomicrsocopy evaluation, inhalation anesthesia with isoflurane is used. Why have you used different protocols?

8. In order to strictly adhere to the three R's principle, why have you not used the 8 rats that were used for photoacoustic validation for histology, immunohistochemistry or Western blot studies?

**********

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.

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Reviewer #1: No

Reviewer #2: Yes: Alberto Ballestín

PLoS One. 2020 Oct 6;15(10):e0239517. doi: 10.1371/journal.pone.0239517.r004

Author response to Decision Letter 1

1 Jun 2020

Review of the manuscript PONE-D-19-21979R1 by Frueh et al.

We have appreciated the fair and constructive comments of the reviewers. Please find our point-by-point reply in the following.

Reply to the comments of reviewer 1

Reviewer comment 1:

In the result section (page 5), “r and t = 900 μm“, and in the figure legend of Fig. 2, “R and T= 900 μm” are not compatible.

We have corrected the corresponding section according to the reviewer’s comment (see page 6, line 151, marked in yellow).

Reviewer comment 2:

On page 6, “ In the IR-120 group, however, we observed a partial loss of the lymph node architecture (Figs. 3F and 3G)”, which particular part was lost?

Our statement was only based on qualitative assessments of lymph node morphology. However, we did not further analyze whether specific parts of the lymph node architecture were lost. Accordingly, we have removed this vague statement from our revised manuscript and now solely focus on the quantitative analysis of lymph node cellularity (see page 6, lines 156-159 and page 12, line 306-312; marked in yellow).

Reviewer comment 3:

In Fig. 4D, the CD 31 staining seems more abundant by compared to the other two groups, which was inconsistent to the results of Fig. 4 E.

The reviewer is correct that the CD31 staining in Fig. 4D looks more abundant. However, the red area in the center of the lymph node is due to local hemorrhages (ie, red stained clusters of erythrocytes). Importantly, microvessels characterized by a CD31+ endothelium are marked with arrowheads for the reader. In addition, the hemorrhages are now marked with separate arrows (see revised Fig. 4D and page 7, line 190, marked in yellow).

Reviewer comment 4:

According to the discussion, authors referenced Gust’s paper indicated “an active role of adipose tissue in driving the inflammatory response after IR injury [26]. The overexpression of stress and inflammatory markers as well as inflammatory cell infiltration of mature adipose tissue following IR injury. But other authors found that severe ischemia is associated with loss of mature adipocytes, which are replaced with new adipocytes derived from resident adipose-derived progenitor cells [27]. “

Since the results showing a higher level of eNOS presented in the perinodal adipose tissue in Fig. 6, there was no significance cell proliferation, apoptosis, no oxidative stress in Fig. 7, which were inconsistent with the results from the reference that the authors provided. How to explain these findings?

We appreciate this important comment. Indeed, it is noteworthy that in our investigation, the adipose tissue was characterized by a less dramatic reaction to IR injury. However, a more thorough look at the referenced animal model by Gust et al. reveals important technical differences when compared to our experimental model. Importantly, Gust et al. used an IR model with magnetic skin compression. In contrast, our IR model exposes the adipose tissue to indirect ischemia without mechanical manipulation. Hence, the less extensive tissue trauma might also result in fewer IR-associated tissue alterations. We now provide this explanation in the discussion section of our revised manuscript (see page 13-14, lines 365-373, marked in yellow).

Reviewer comment 5:

The results indicated an increasing level of VEGF-A after ischemia condition (Fig. 5). The results were not consistent with that in Fig. 4, in which the micro-vessel density was decreased after ischemia condition.

From a biological point of view, this finding is not surprising because IR-related tissue damage leads to the loss of microvascular structures and local hypoxia. Consequently, angiogenic growth factors such as VEGF-A are upregulated, promoting angiogenic neovessel growth. However, angiogenesis is a time-consuming process with a slow growth rate of new microvessels of 5 µm per hour. Hence, 24 hours of reperfusion may be too short to demonstrate a subsequent increased vascularization. This information is now provided in the revised discussion of our manuscript (see page 13, lines 345-352 and page 23, lines 627-628; marked in yellow).

New reference

Utzinger U, Baggett B, Weiss JA, Hoying JB, Edgar LT. Large-scale time series microscopy of neovessel growth during angiogenesis. Angiogenesis. 2015;18:219-232.

Reviewer comment 6:

The staining of CD31 in Fig. 4D was not compatible with Figs. 4E and 4F.

See reply to comment 4.

Reply to the comments of reviewer 2

Reviewer comment 1:

Indicate in the title, in the abstract and when the objective of the study is stated at the end of the introduction, that the main goal is to investigate the “immediate” or “initial” changes at the cellular and molecular level after performing lymph node transfers, since the study ends at 24h postop. Findings may be interpreted as biomarkers to identify the initial phase of I/R injury in lymph nodes and perinodal tissue.

We thank the reviewer for this important comment. According to his suggestion, we now state in the title, abstract and introduction that our study investigates the short-term molecular and cellular effects of I/R on VLN (see page 1, line 3 and page 2, line 41 and page 4, line 99 and page 11, line 296, marked in yellow).

Reviewer comment 2:

The abstract does not indicate which animal model or which lymph nodes are studied, nor the number of animals, nor their distribution in the study groups. Please modify it accordingly.

According to this comment of the reviewer, we now provide the total number of animals used in the study as well as more details on the animal model in the abstract (see page 2, lines 42-44, marked in yellow).

However, to enhance the readability of the manuscript, we prefer not to mention the distribution of the animals in the study groups in the abstract. In fact, this essential information is already provided in two sections of our manuscript (ie, experimental protocol and figure legends).

Reviewer comment 3:

Please modify the image Figure 1.B as it is not clearly seen.

We have modified the figure including a more detailed explanation in the corresponding figure legend (see revised Figs. 1B and 1C and corresponding figure legend, page 4, lines 119-124, marked in yellow).

Reviewer comment 4:

In the discussion please focus more on your actual results and their interpretation. This section partially sounds like an introduction. You may move parts to the introduction if needed.

According to this comment of the reviewer, we have moved parts of the discussion to the introduction and focused more on our results (see page 3, lines 87-94 and pages 11-14, lines 306-385; marked in yellow).

Reviewer comment 5:

In the final paragraph of the discussion (conclusion) try to be more specific and summarize the significant results you have obtained in this study. This will help other researchers and readers to get the most relevant results of this study.

According to this comment, we have modified the last paragraph of the discussion in our revised manuscript (see page 14-15, lines 398-406, marked in yellow).

Reviewer comment 6:

It is important that readers keep in mind the limitations of this study. Include a summary of the main ones and explain them in the discussion. Examples:

- There are numerous growth factors that were not included in the analysis, in this sense, we should not exclude the importance of other biomarkers.

- Vascularized lymph node transfers are performed using microsurgical anastomoses, however in this study, VLNTs have been simulated by clamping the vessels.

- The current literature indicates that the critical ischemia time in lymph node transfers is 4h, however in this study a shorter ischemic insult has been studied.

We thank the reviewer for this comment. According to his suggestion, we have included a novel paragraph on the limitations of our study in the discussion section of our revised manuscript (see page 14, lines 386-397, marked in yellow).

Reviewer comment 7:

In the Material and Methods section, it is indicated that an injectable anesthetic protocol (Ketamine and xylazine) is used during surgery, but during the ultrasound, photoacoustic imaging and stereomicroscopy evaluation, inhalation anesthesia with isoflurane is used. Why have you used different protocols?

The reviewer is correct that flap dissection was performed under intraperitoneal anesthesia using ketamine and xylazine. After transfer and fixation of the animals in the ultrasound/photoacoustic imaging system, maintenance of anesthesia was performed using isoflurane inhalation under continuous monitoring of heart and breathing rate as well as body temperature. On the one hand, inhalation anesthesia is suitable to precisely control the depth of anesthesia. On the other, the animal is fixed in a complex imaging system, which renders intraperitoneal injection of anesthetics cumbersome and a source of bias due to animal manipulation, particularly during photoacoustic imaging.

Reviewer comment 8

In order to strictly adhere to the three R's principle, why have you not used the 8 rats that were used for photoacoustic validation for histology, immunohistochemistry or Western blot studies?

We thank the reviewer for this important comment. However, it can be answered by maximal standardization of the perioperative protocol. Including these rats in the groups for histology, immunohistochemistry or Western blot analyses would not only have resulted in a different protocol (ie, transfer and delay for photoacoustic imaging), it would also be biased by different anesthesia medications as correctly stated by the reviewer in comment 7. Finally, we performed photoacoustic imaging for the IR-45 and IR-120 groups but not for the control group. Hence, inclusion of these animals for subsequent histological, immunohistochemical or Western blot analyses would have also resulted in uneven numbers of the experimental groups.

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PLoS One. doi: 10.1371/journal.pone.0239517.r005

Decision Letter 2

Zhejun Cai

14 Aug 2020

PONE-D-19-21979R2

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

PLOS ONE

Dear Dr. Frueh,

Please submit your revised manuscript by Sep 28 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Zhejun Cai, M.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

6. Review Comments to the Author

Reviewer #1: This is the revision manuscript. Authors tried to answers most of reviewer’s comments.

Many inconsistent statements had been corrected. The scientific concepts are clearer in this revision manuscript. There are few comments as follows.

a. In Fig 3, any difference on LYVE expression, since it’s the marker for lymphatic network.

b. In Fig.4, it will be more convicting when the images of CD31staining in perinodal adipose are provided.

Reviewer #2: Authors have adequately addressed the comments raised in the previous round of review. The authors have refined the article by specifying that the objective was to study "immediate" or "initial" changes at the cellular and molecular level after performing lymph node transfers. In addition, both the introduction and the discussion have been improved, as well as the main limitations of the preclinical study have been included.

It is a scientific paper with a good research set-up which is well explained as well as conducted. It describes the results of a preclinical study analyzing the immediate molecular and cellular effects after ischemia-reperfusion injury on vascularized lymph node transfers. For this study the authors have used a rat model of vascularized lymph node flap pedicled on the lateral thoracic vessels. The article analyzes various lymphangiogenic growth factors, as well as it evaluates the morphology, proliferation, apoptosis, and infiltration of immune cells in the lymph nodes and perinodal adipose tissue in this murine model. The study demonstrates that vascularized lymph node flaps are prone to ischemia reperfusion injury, being associated with a significant reduction of cellularity and vascularization of lymph nodes, as well as apoptotic cell death and immune cell infiltration.

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Oct 6;15(10):e0239517. doi: 10.1371/journal.pone.0239517.r006

Author response to Decision Letter 2

20 Aug 2020

We have appreciated the fair and constructive comments of the reviewer. Please find our point-by-point reply in the following.

Reply to the comments of reviewer 1

Reviewer comment 1

In Fig 3, any difference on LYVE expression, since it’s the marker for lymphatic network.

We thank the reviewer for this comment. However, we have already discussed this point in the first review round and hereby provide our original reply:

“The reviewer is correct that LYVE-1 staining in this part of our investigation was only used for descriptive purposes and not for a quantitative analysis of lymphatic vessel density. In this context it should be noted that the expression of LYVE-1 in the lymphatic sinuses of lymph nodes is not limited to lymphatic endothelial cells but is also observed in other cell types, such as reticular cells (Bai et al., 2011). Consequently, a quantification of the lymphatic vascular network (ie, the sinuses) within individual lymph nodes is not appropriate using LYVE-1 as marker.”

Reference

Reviewer comment 2

In Fig.4, it will be more convicting when the images of CD31staining in perinodal adipose are provided.

According to this comment of the reviewer, we now provide immunofluorescent images illustrating the CD31+ microvascular network in the perinodal adipose tissue in Fig. 4 (see revised Fig. 4 as well as the corresponding figure legend on page 7, lines 187-190 in the revised version of our manuscript, marked in yellow).

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PLoS One. doi: 10.1371/journal.pone.0239517.r007

Decision Letter 3

Zhejun Cai

9 Sep 2020

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

PONE-D-19-21979R3

Dear Dr. Frueh,

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.

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Kind regards,

Zhejun Cai, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

PLoS One. doi: 10.1371/journal.pone.0239517.r008

Acceptance letter

Zhejun Cai

25 Sep 2020

PONE-D-19-21979R3

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

Dear Dr. Frueh:

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.

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

Kind regards,

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on behalf of

Dr. Zhejun Cai

Academic Editor

PLOS ONE

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats

Florian S Frueh

Bijan Jelvani

Claudia Scheuer

Christina Körbel

Bong-Sung Kim

Pietro Giovanoli

Nicole Lindenblatt

Yves Harder

Emmanuel Ampofo

Michael D Menger

Matthias W Laschke

Roles

Abstract

Introduction

Results

Animal model

Fig 1. The axillary VLN flap model.

Validation of animal model

Fig 2. Animal model validation.

Morphology of VLN flaps

Fig 3. Structural alterations of VLN flaps after IR injury.

Microvascular network analysis

Fig 4. Microvessel density of VLN flaps after IR injury.

Expression of lymphangiogenic and angiogenic growth factors

Fig 5. Growth factor and NOS expression of VLN flaps after IR injury.

Expression of iNOS, eNOS and p-eNOS

Oxidative stress, cell proliferation, cell apoptosis

Fig 6. Immunohistochemical analyses of lymph nodes after IR injury.

Fig 7. Immunohistochemical analyses of adipose tissue after IR injury.

Immune cell infiltration

Discussion

Material and methods

Animals

Surgery

Ultrasound, photoacoustic imaging and stereomicroscopy

Western blot

Histology and immunohistochemistry

Experimental protocol

Statistics

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ferenc Gallyas, Jr

Roles

Author response to Decision Letter 0

Decision Letter 1

Jianhong Zhou

Roles

Author response to Decision Letter 1

Decision Letter 2

Zhejun Cai

Roles

Author response to Decision Letter 2

Decision Letter 3

Zhejun Cai

Roles

Acceptance letter

Zhejun Cai

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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