Animal models in surgical lymphedema research—a systematic review

Accepted Manuscript Animal Models in Surgical Lymphedema Research - A Systematic Review Florian S. Frueh, MD, Epameinondas Gousopoulos, MD MSc, Farid Rezaeian, MD, Michael D. Menger, MD, Nicole Lindenblatt, MD, Pietro Giovanoli, MD PII:

S0022-4804(15)00750-7

DOI:

10.1016/j.jss.2015.07.005

Reference:

YJSRE 13457

To appear in:

Journal of Surgical Research

Received Date: 23 April 2015 Revised Date:

24 June 2015

Accepted Date: 2 July 2015

Please cite this article as: Frueh FS, Gousopoulos E, Rezaeian F, Menger MD, Lindenblatt N, Giovanoli P, Animal Models in Surgical Lymphedema Research - A Systematic Review, Journal of Surgical Research (2015), doi: 10.1016/j.jss.2015.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

ACCEPTED MANUSCRIPT

Revised, 26.06.2015

1 2

Animal Models in Surgical Lymphedema Research

3

A Systematic Review

RI PT

4 Florian S. Frueh MD1, Epameinondas Gousopoulos MD MSc2, Farid Rezaeian MD1,

6

Michael D. Menger MD3, Nicole Lindenblatt MD1*, Pietro Giovanoli MD1*

7 8

* These authors contributed equally to this work

10

M AN U

9 1

Division of Plastic Surgery and Hand Surgery, University Hospital Zurich, 8091 Zurich, Switzerland

2

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland

3

Institute for Clinical and Experimental Surgery, University of Saarland, 66424 Homburg/Saar, Germany

11 12

15

TE D

13 14

Corresponding Author

17

Nicole Lindenblatt MD

18

Division of Plastic Surgery and Hand Surgery

19

University Hospital Zurich

20

Raemistrasse 100

21

CH-8091 Zurich

22

Switzerland

23

E-Mail: [email protected]

24

Phone: +41 44 255 11 11

26

AC C

EP

16

25

SC

5

2

ACCEPTED MANUSCRIPT 27

Authors' Contribution

28

Study design and data assessment: FSF, FR & NL. Data analysis: FSF & EG. Wrote the manuscript:

29

FSF, EG, FR & MDM. Critical manuscript revision: NL, MDM & PG.

30

Conflict of Interest

32

The authors report no proprietary or commercial interest in any product mentioned or concept

33

discussed in this article.

34

Running Head

36

Animal models in surgical lymphedema research

37 38

Word Count

39

Manuscript (without references): 4431

43 44 45 46 47 48 49 50

EP

42

AC C

41

TE D

40

M AN U

35

SC

RI PT

31

3

ACCEPTED MANUSCRIPT

ABSTRACT

52

Background. Chronic secondary lymphedema is a well-known complication in oncologic surgery.

53

Autologous lymph node transplantation, lymphovenous anastomosis and other lymphatic surgeries

54

have been developed in the last decades with rising clinical application. Animal models to explore the

55

pathophysiology of lymphedema as well as microsurgical interventions have reached great popularity,

56

although the induction of stable lymphedema in animals is still challenging. The aim of this review

57

was to systematically assess lymphedema animal models and their potential use to study surgical

58

interventions.

59

Materials and Methods. A systematic review according to the PRISMA guidelines was performed

60

without time or language restriction. Studies describing new or partially new models were included in

61

chronological order. Models for primary and secondary lymphedema were assessed and their potential

62

for surgical procedures was evaluated.

63

Results. The systematic search yielded 8590 discrete articles. Of 180 articles included on basis of title,

64

83 were excluded after abstract review. Ninety-seven were included in the final analysis with 24 key

65

articles.

66

Conclusions. No animal model is perfect and many models show spontaneous lymphedema resolution.

67

The rodent limb appears to be the most eligible animal model for experimental reconstruction of the

68

lymphatic function as it is well accessible for vascularized tissue transfer. There is a need for

69

standardized parameters in experimental lymphedema quantification. Also, more permanent models to

70

study the effect of free vascularized lymph node transfer are needed.

SC

M AN U

TE D

EP

AC C

71

RI PT

51

72

Keywords

73

Animal model; Lymphatic system; Lymphedema; Lymphoedema; Lymph node transplantation

74 75 76 77

4

ACCEPTED MANUSCRIPT 78 79

1. INTRODUCTION Beside to the blood vasculature, the lymphatic system significantly contributes to the regulation of vital functions in the human body, including the control of tissue pressure, immune

81

surveillance and intestinal dietary fat absorption.1 It consists of lymphatic capillaries, pre-collecting

82

vessels and collecting lymphatic trunks, which form a 3-dimensional network with interposed lymph

83

nodes. The lymph nodes represent the "immunologic center" of the lymphatic network and are

84

essential for the initiation of immune responses. Furthermore, the lymphatic system is involved in

85

several pathological processes such as lymphedema, cancer dissemination and inflammatory

86

disorders.1, 2 Failure of the lymphatic system to efficiently drain the extravasated fluid leads to

87

accumulation of lymph fluid in the interstitial tissue, causing lymphedema. Its chronic form is

88

characterized by swelling, tissue fibrosis, adipose tissue accumulation and immune cell infiltration.

SC

M AN U

89

RI PT

80

Lymphedema can be classified based on its cause. Primary lymphedema, further classified by the age of onset as hereditary, praecox or tarda, is a rare disease with an estimated prevalence rate of

91

1.15/100'000 subjects in North America.3 It originates from causal mutations affecting lymphatic

92

development and usually involves the lower extremities of female patients. Milroy Syndrome

93

(VEGFR-3 encoding gene), Meige Syndrome (mutation unknown), Lymphedema-Distichiasis (FOXC2

94

on chromosome 16) and Yellow Nail Syndrome are hereditary diseases associated with primary

95

lymphedema.4, 5

EP

96

TE D

90

In contrast, secondary (acquired) lymphedema represents a common complication after operative procedures in oncologic surgery, such as axillary or inguinal lymph node dissection in the

98

context of breast cancer or melanoma treatment.6 Up to 30% of women treated for breast cancer and

99

around 20% of patients after inguinal lymph node dissection for melanoma develop lymphedema.7, 8

AC C

97

100

Although the extent of surgery correlates with the risk of developing permanent lymphedema9,

101

secondary lymphedema has also been described following sentinel lymph node biopsy.7 In addition,

102

radiation, infectious diseases (i.e. lymphatic filariasis) or chronic inflammation may also cause

103

secondary lymphedema. According to the 2014 WHO report

104

(http://www.who.int/mediacentre/factsheets/fs102/en/index.html) over 15 million people are suffering

5

ACCEPTED MANUSCRIPT 105

from lymphedema due to lymphatic filariasis, making it the most important etiology of secondary

106

lymphedema in developing countries. However, in industrialized countries, cancer treatment as

107

outlined above is the most frequent cause of secondary lymphedema.

108

Despite advances in all fields of surgery, physiotherapy (i.e. lymph drainage, compressive bandages) remains the standard symptomatic treatment for both primary and secondary lymphedema.

110

However, as the disease can be caused by surgical intervention, surgical treatment has been discussed

111

over decades. Ablative and invasive surgery is not applied anymore today due to high morbidity and

112

bad cosmetic outcome.10, 11 Since super-microsurgical techniques (i.e. the possibility of lymph vessel

113

anastomosis and vascularized lymph node transfer) have become available, lymphovenous

114

anastomosis (LVA), lymphatic vessel transplantation and autologous lymph node transplantation

115

(ALNT) exhibit encouraging results in reconstructive microsurgery.12, 13 Moreover, the progress in the

116

surgical field coupled with the advances in tissue-engineering render the transplantation of engineered

117

lymph nodes and lymphatic vessels as promising approaches to restore lymphatic function for the

118

treatment of lymphedema.14, 15

M AN U

SC

RI PT

109

Because lymphedema is a complex disease involving a multitude of tissue components, the

120

development of in vitro systems to dissect its pathophysiology is difficult. Hence, the use of animal

121

models is indispensable. Various pre-clinical models, ranging from dogs to rodents, have been

122

developed to investigate the underlying biology of lymphedema and explore therapeutic interventions.

123

In the light of evolving microsurgical procedures to treat lymphedema, there is a lack of evidence for

124

their clinical efficacy, confining surgical management of lymphedema as case series and anecdotal

125

reports.16 We understand that there is a need for reliable experimental animal models, reproducibly

126

replicating the disease pathophysiology and potential curative treatments to refine these techniques.

127

Therefore, it has been the aim of this review to systematically assess the different lymphedema animal

128

models, analyzing the accessibility for the exploration of surgery-based treatment options.

129 130 131 132

AC C

EP

TE D

119

6

ACCEPTED MANUSCRIPT 133

2. MATERIALS AND METHODS

134

2.1 Systematic Review

135

A review protocol was designed in advance and has been registered on http://www.dcn.ed.ac.uk/camarades//research.html#protocols. We performed a systematic review in

137

accordance to the PRISMA guidelines.17 Details of the review process are shown in Table 1. In brief,

138

search terms were focused on animal models and lymphedema, excluding clinical trials, reviews and

139

non-related disease models. Each study was verified for the relevance to the topic and from the

140

surgeon's point of view (required microsurgical skills/equipment, Table 2).

141

The systematic search yielded 8590 discrete articles. Of 180 articles included on basis of title, 83 were

142

excluded after abstract review. Ninety-seven were included in the final analysis with 24 "key articles"

143

describing new or partially new models. The detailed selection process is shown in Fig. 1.

M AN U

SC

RI PT

136

144

146

3. RESULTS

The results have been categorized based on the animal model to enable categorized

TE D

145

comparisons. Due to the heterogeneity of the studies, including the techniques to induce lymphedema,

148

the therapeutic approaches attempted and the surgical relevance, the results have been organized into

149

table format facilitating visual paralleling.

151 152

3.1 Canine Models (Table 3)

AC C

150

EP

147

Danese et al18 performed a surgical resection of the deep femoral lymphatics. A two-inch

153

circumferential defect was produced. Perioperative mortality rate was high with 25 %. Lymphedema

154

was assessed by clinical observations and lymphangiography. Circumferential excision alone resulted

155

in moderate and spontaneously receding lymphedema. Lymphangiography revealed dilation of

156

lymphatic vessels and loss of valvular competence.

157

Olszewski et al19 achieved stable secondary lymphedema in mongrel dogs. They performed a

158

circular excision of 20 mm width in the proximal thigh. Leaving the muscles intact, they resected 40

159

mm of femoral lymphatics and skeletonized the femoral vessels. Additionally, excision of the popliteal

7

ACCEPTED MANUSCRIPT 160

lymph node was performed. 35 % of the animals developed permanent elephantiatic lymphedema 7 to

161

10 months after surgery. Lymphography showed dilated and tortuous lymphatics and signs of

162

lymphatic regeneration through the scar. Pflug et al20 produced lymphedema in seven female greyhounds. They isolated the three main

164

lymphatics at the calf and paw. Intralymphatic injection of neoprene latex was performed. One month

165

postoperatively, all dogs developed gross edema. At 5 months, four animals showed edematous limbs.

166

RI PT

163

Clodius et al21 described a radical circular excision of thigh lymphatics in dogs. An Etheron sponge was used to fill the surgical gap. Etheron consists of di-isocyanate of polyether and allows

168

ingrowth of fibrous tissue only. In a second group animals were treated in the same way except

169

preserving epifascial lymphatic flow over anterior and posterior skin bridges. Animals with intact skin

170

bridges developed reversible initial edema, whereas the other dogs died within three weeks following

171

massive protein loss. Serial lymphography showed reverse flow from subfascial to dilated epifascial

172

lymphatics.

M AN U

173

SC

167

Das et al22 combined surgery and radiotherapy in female dogs of mixed breeds. Groin block excision with resection and ligation of deep lymphatics was performed. Animals of a first group (n=8)

175

were operated followed by radiation with 1500 rads orthovoltage three to six weeks later. Animals of a

176

second group (n=15) were irradiated before surgery. Femoral vessels were covered with distally

177

pedicled gracilis and sartorius flaps. Mortality rate was high with 30 %. The combination radiation

178

followed by surgery was very effective with 100 % chronic lymphedema, whereas surgery followed

179

by radiation resulted in chronic lymphedema of only two out of eight animals. Some dogs were treated

180

with LVA. However, no results were reported.

EP

AC C

181

TE D

174

Chen et al23 confirmed the importance of preoperative radiation. They irradiated mongrel dogs

182

with 1200 rads at the level of the knee. Within a month, circumferential excision of skin and

183

subcutaneous tissue with ligation of main lymphatics was performed. Three months later, all deep

184

lymphatics and contents of the popliteal fossa were removed. Twelve out of 14 dogs developed

185

chronic lymphedema, defined as at least 15 % increase in limb circumference. In a control group, ten

186

animals received surgical procedure alone, which did not result in reliable lymphedema induction.

187

8

ACCEPTED MANUSCRIPT 188

3.2 Rabbit, Sheep and Pig Models (Table 4) Casley-Smith et al24 produced lymphedema in the rabbit ear of 11 animals with a dorsal, 4 cm

190

wide excision of skin, subcutaneous tissue and perichondrium sparing the anterior auricular artery and

191

vein. Four animals were daily treated with intraperitoneally injected benzopyrones. No information is

192

given on how many of the 11 animals developed lymphedema. Six weeks postoperatively, samples

193

from operated and healthy ears were examined with electron microscopy. Edematous tissue and

194

lymphatics of animals receiving benzopyrones showed lesser edema and lower protein concentration.

195

The authors concluded that benzopyrones were effective due to induction of macrophage activity. Huang et al25 compared radical (n=25) and partial (n=25) lymphatic block in the ears of

SC

196

RI PT

189

Japanese rabbits. Partial surgical block consisted of preserving two to four central lymphatics along

198

the central neurovascular bundle. 94 % of animals developed lymphedema. Ear thickness was

199

significantly increased in both groups for 30 days. Both radical and partial lymphatic block resulted in

200

stable lymphedema production.

201

M AN U

197

Dominici et al26 produced secondary lymphedema in New Zealand White rabbit ears. One group of animals underwent surgical interruption of lymph vessels. A second group was treated with

203

direct electrocoagulation of the skin and subcutaneous tissue. During a follow-up period of 30 days

204

both procedures resulted in conspicuous edema. The authors indicated that LVA was performed,

205

however, without reporting results. They concluded that electrocoagulation was a fast and easy

206

method to produce acute lymphedema in the rabbit ear.

EP

TE D

202

Szuba et al27 examined the impact of VEGF-C on secondary lymphedema in eight rabbit ears.

208

Edema was induced modifying the technique described by Huang et al25. Two of the 18 rabbits failed

209

to develop lymphedema. After complete reepithelialisation 100 µg of recombinant human VEGF-C

210

were injected intradermally at the site of lymphatic resection. After a further eight-day period

211

immunohistochemistry revealed an increased lymphatic vascularity and a reduced tissue

212

hypercellularity compared to saline-treated lymphedema ears.

AC C

207

213

Tobbia et al28 described the induction of lymphedema in 41 sheep performing popliteal lymph

214

node excision. Limb circumference was increased in all animals with maximum response at day three

9

ACCEPTED MANUSCRIPT after surgery (average increase 33.8 %). Over time, lymphedema was reversible with complete

216

remission in three sheep. Lymphatic transport capacity was assessed with prenodal injection of

217

radiolabeled human serum albumin (125I-HSA). Twelve and 16 weeks after lymph node removal,

218

lymphatic function was restored to 80 % compared to healthy animals. These findings correspond to

219

the results of others, reporting lymphatic regeneration after popliteal lymph node removal in sheep.29

RI PT

215

Lähteenvuo et al30 produced acute lymphatic damage by resecting all afferent and efferent

221

lymphatics to an inguinal lymph node in domestic pigs. They preserved the vascular pedicle of the

222

node, thus mimicking autologous lymph node transplantation. Particles of adenoviral vectors encoding

223

VEGF-C and VEGF-D were subcapsularly injected into the node. Lymphangiography was performed

224

2 months after gene transfer and showed that the application of growth factors induced lymphatic

225

regeneration.

M AN U

226

SC

220

Honkonen et al31 evaluated the efficacy and the potential adverse effects of intra- and perinodal lymphatic growth factor treatment in the same pig model. They induced acute lymphatic

228

damage and the analysis of intra- and perinodal VEGF-C administration showed a higher number of

229

lymphatic vessels and more frequent connections to the lymph nodes compared to control animals.

230

Perinodal and intranodal growth factor injection showed comparable results.

TE D

227

231

3.3. Rodent Models (Table 5, Fig. 2)

233

3.3.1 Rat Models

EP

232

Wang et al32 produced secondary lymphedema in rat hind limbs. After a circumferential

235

incision, they resected the main lymphatic trunk and the popliteal lymph node. Skin edges were

236

sutured to the muscle to exclude collateral flow. The contralateral hind limb was used as control. After

237

six months, 64 % of the rats showed some degree of lymphedema. The mortality rate was high with

238

severe wound infection representing the most frequent cause of death.

239

AC C

234

Huang et al33 described a model of secondary lymphedema in albino rat hind limbs without

240

resecting all lymphatics. They incised soft tissue in the middle portion of the limbs preserving

241

saphenous vessels and the main lymphatics. Rats were sacrificed at different stages and

10

ACCEPTED MANUSCRIPT 242

histopathological analysis was performed. Over a 26-weeks period following surgery the increase of

243

the circumference ranged constantly between 30 % and 40 %. Lymphatic contraction showed

244

increased activity until week eight. Afterwards, decompensation of lymphatic function was observed.

245

Kanter et al34 achieved stable secondary lymphedema in Sprague-Dawley rats combining preoperative radiation (4500 rads) with circumferential division of thigh lymphatics. Lymphedema

247

was quantified by measurement of limb circumference and Tc99 Sb2S3 lymphoscintigraphy.

248

Lymphedema persisted for up to nine months.

RI PT

246

Levine et al35 described parasternal lymphedema in rats after intraperitoneal injection of

250

glutaraldehyde-treated rat red blood cells. The presence of lymphedema was assessed microscopically.

251

Acute lymphedema was inducible with a maximum after six hours up to two days following

252

inoculation. Four to seven days after the procedure, only dilated lymphatics and slight lymphedema

253

were still present. Lymphedema was localized within the compartment between the transversus

254

thoracis and the intercostal muscles. Main disadvantage of this model is the short availability of

255

lymphedema.

M AN U

Kawahira et al36 compared different surgical treatments for secondary lymphedema in adult

TE D

256

SC

249

male Sprague-Dawley rats. Lymphedema was produced by surgical dissection of all hind limb

258

lymphatics. A conventional Kinmonth procedure (left pararectal approach) was performed in animals

259

of group one. The Kinmonth operation37 consists of suturing a vascularized bowel loop to the subcutis

260

of the mid-thigh. Animals of group two underwent a modified Kinmonth procedure with additional

261

harvesting of a part of the greater omentum. In animals of group three, ALNT was performed with

262

lymph node capsulo-venous anastomosis to the femoral vein. Axillary lymph nodes were harvested for

263

transplantation. The modified Kinmonth procedure has been shown to reduce lymphedema most

264

effectively.

265

AC C

EP

257

Lee-Donaldson et al38 reported induction of secondary lymphedema in Wistar rats. They

266

performed microsurgical ablation of groin lymph nodes and lymphatics and/or groin radiation with

267

4500 rads. Radiation alone did not produce any significant edema. Radiation before surgery and

268

radiation after surgery resulted in permanent lymphedema, whereas rats treated with surgery alone

11

ACCEPTED MANUSCRIPT 269

developed only transient limb edema. In this model stable secondary lymphedema could be

270

accomplished for up to 100 days.

271 272

3.3.2 Mouse Models Slavin et al39 examined the effect of a pedicled myocutaneous rectus abdominis flap on

274

lymphedema in rat and mouse-tails. The tails of Fisher rats were incised circumferentially with

275

ligation of the two deep lymphatic trunks. In 11 animals, a myocutanous rectus abdominis was

276

transposed close to the lymphatic ligation. The same procedure was performed in two groups of

277

female nude mice with the exception that the lymphatic channels were cauterized rather than ligated.

278

Tail diameter, lymphoscintigraphy (Tc-99m-An-sulfur) and fluorescent microlymphangiography were

279

compared with control groups. The study showed that flap transfer significantly reduced the tail

280

diameter. Lymphoscintigraphy at three weeks after surgery showed that lymphatic continuity after

281

ligation and flap was indistinguishable from that in the control groups.

SC

M AN U

282

RI PT

273

Tabibiazar et al40 used the same mouse-tail model and identified inflammation as a main contributor in lymphedema pathophysiology. Tail volume measurements, in vivo functional imaging

284

of immune trafficking, lymphoscintigraphy and histopathology were used to describe the pathologic

285

characteristics of the disease. Molecular characterization of the affected tissue using a microarray

286

indicated profound inflammation, immune response, fibrotic changes and wound healing profile.

EP

287

TE D

283

Karkkainen et al41 described a model for gene therapy of primary lymphedema in two mouse strains. The Chy phenotype showed chylous fluid in the abdomen and swelling of the feet. Inactivation

289

of VEGFR-3 was achieved by ethylnitrosourea-mediated mutagenesis. Lymphedema was quantified

290

with MRI comparing the Chy phenotype with wild-type mice. Immunohistochemistry showed

291

enlarged cutaneous lymphatic vessels in the Chy mice, corresponding to findings in patients with

292

Milroy Syndrome. The authors performed gene therapy with intradermal injection of virus-mediated

293

VEGF-C (ears), which resulted in functional cutaneous lymphatics three to seven weeks after

294

application.

295 296

AC C

288

Tammela et al42 combined lymph node transfer and local lymphatic growth factor treatment in a newly established secondary lymphedema mouse model. They induced lymphedema in NMRI nude

12

ACCEPTED MANUSCRIPT 297

mice through resection of the axillary lymph nodes and lymphatic vessels. Local growth factor

298

treatment (adenoviral VEGF-C or VEGF-D) was applied to the axillary wound. One week later, lymph

299

node fragments without microvascular anastomosis were allografted. The application of VEGF-C

300

improved the regeneration of the lymphatic function. Oashi et al43 introduced a new model of hind limb lymphedema in mice. After radiation of the

RI PT

301

left groin with 30 gray, they resected the deep lymphatics and subiliacal and popliteal lymph nodes.

303

Limb volume and indocyanine green (ICG) fluorescence were used to measure lymphedema. One

304

week after surgery, ICG measurements revealed a dramatic disappearance of major lymphatic trunks.

305

All surviving animals developed lymphedema.

307 308

M AN U

306

SC

302

4. DISCUSSION

Animal models are indispensable in translational research, when evaluating the underlying pathobiology and therapeutic modalities to predict human outcomes. Different models can be useful

310

for different approaches and the selection of the model should be based on the objectives and designed

311

methodology. Lymphedema is a complex disease mainly characterized by lymphatic insufficiency,

312

which has to be imitated in the animal model, leading to the expected phenotype. The anatomic

313

structures, the nature of the study, the techniques involved and interventions attempted co-define,

314

among others, the experimental model to be used.

316 317

EP

4.1 Large Animal Models

AC C

315

TE D

309

Early on in lymphatic research in the 1930s, Homans et al44 induced chronic lymphedema in

318

dogs' hind limbs using a complicated procedure involving lymphatic ligation and intralymphatic

319

injection of a sclerosing solution. In the following years, many attempts have been undertaken to

320

simplify edema induction. In these studies it was recognized that radical lymphatic excision21 and pre-

321

or postoperative radiation22, 23 are important to achieve an adequate lymphedema. Several authors have

322

performed LVA in dog models with secondary lymphedema. Whereas end-to-end anastomoses were

323

mostly found occluded after a few weeks, end-to-side anastomoses showed patency rates of up to 80

13

ACCEPTED MANUSCRIPT

% after six months.45-47 Kinjo et al48 recommended additional distal ligation of the venous segment and

325

external valvuloplasty proximal to the anastomosis. Baumeister et al49 considered LVA insufficient for

326

the treatment of lymphedema and proposed the transplantation of lymph vessels. Vascularized ALNT

327

to treat lymphedema was performed by Chen et al.50 They showed progressive edema reduction in ten

328

animals. Lymphaticolymphatic anastomosis did not improve the results compared to spontaneous

329

reconnection. However, mortality rates of the animals were high. Moreover, large animals, such as

330

dogs, sheep and pigs, are both expensive to handle and breed. Due to ethical concerns, the complexity

331

of lymphedema induction and the long latency until chronic lymphedema develops the dog model is of

332

historical value.51 Overall, large animal models require basic microsurgical skills (preparation of

333

lymphatics) and have been mostly replaced by the rodent models for the above-mentioned reasons.

SC

RI PT

324

The sheep model described by Tobbia et al28 is an interesting alternative with 100 % success

335

rate, but edema declination over time. The authors emphasized the "human-sized" perspective of the

336

sheep model, which offers the possibility of disrupting the lymphatic drainage of a whole limb by a

337

single lymph node excision. Vascularized ALNT was associated with a significantly greater

338

improvement of lymphatic function than avascular transplantation.52

TE D

339

M AN U

334

The pig model used by the Finnish group30,31 comes along with similar advantages as the sheep model (animal size, hydrostatic conditions). Limitations include different lymphatic anatomy of

341

the inguinal region.30 Next to this, the model was designed for acute lymphatic damage and not for

342

chronic lymphedema, which leaves its significance for experimental lymphedema research undefined.

344 345

AC C

343

EP

340

4.2 Rabbit Model

The rabbit ear represents a safe and reliable lymphedema model. Standard microsurgical

346

dissection is required to isolate the lymphatics at the base of the ear. The main risk in the rabbit model

347

is cartilage necrosis following perichondrium excision. Another disadvantage of the rabbit ear model

348

might be that it is not sufficiently accessible for microsurgical reconstructive treatment of

349

lymphedema with vascularized tissue transfer, disabling a surgical treatment approach.

350

14

ACCEPTED MANUSCRIPT 351 352

4.3 Rodent Models After the introduction of the rat hind limb as an easily accessible, cost-effective and reliable

353

lymphedema model,32, 33 the use of rodents has gained great popularity. The combination of radiation

354

and surgery has proven to induce most effectively a stable lymphedema in both rats and mice.34, 38, 43 The rodent tail model represents another lymphedema model, reported by Slavin et al.39

356

Compared to the hind limb model, anatomy and surgical technique are simple and well reproducible. It

357

has been used for research of surgical lymphedema treatment, gene therapy and molecular aspects of

358

lymphangiogenesis.39, 51, 53, 54 However, the exact surgical technique may significantly vary, resulting

359

in fluctuations in duration and robustness of the developed lymphedema. Uzarski et al55 and Kimura et

360

al56 maintained the integrity of the collecting lymphatic vessels after removal of a circumferential

361

piece of skin 2 mm from the tail basis. In contrary, both Slavin et al39 and Rutkowski et al54 cauterized

362

the underlying lymphatic vessels, whereas Clavin et al57 ligated them.

M AN U

SC

RI PT

355

So far the mouse-tail model has been mainly utilized to unravel the underlying mechanisms of

364

lymphedema development and its use in surgical approaches has been limited. To that end, Tabibiazar

365

et al40, using a microarray, highlighted the importance of inflammation in lymphedema development

366

and the key pathways involved in the disease`s progression. The results offer insights into

367

lymphedema`s molecular background and might be useful suggesting relevant potential therapeutic

368

applications.

EP

TE D

363

The rodent models represent an easily available microsurgical training opportunity, yet

370

challenging due to the animal size and the advanced microsurgical skills needed. Rodent models have

371

been used to exploit the therapeutic potential of ALNT, including both lymph node fragment

372

transplantation with the addition of VEGF-C58 and vascularized ALNT.59, 60 Lymphatic function after

373

transplantation has been demonstrated.59

374

AC C

369

Due to the availability of the necessary molecular biology tools in mice, including transgene

375

animals and antibodies, they have been utilized in molecular studies. The primary lymphedema

376

models by Karkkainen et al41, Kriedermann et al61 and Makinen et al62 are of outmost value for the

377

understanding of lymphedema pathophysiology. As the animals show congenital dysfunctional

15

ACCEPTED MANUSCRIPT 378

lymphatic capillaries, surgery has minimal to no effect on these entities, making them less interesting

379

for experimental surgical research.

380

382

4.4 Definition of Experimental Lymphedema One major problem of experimental lymphatic surgery is the lack of standardized parameters

RI PT

381

for objective lymphedema assessment. There is no accepted standard for an animal model and no

384

common definition of lymphedema has been used in the described studies. It is debatable whether we

385

can compare the results of animals with lymphedema if they show an increase of limb circumference

386

of 5% versus 20%.

In order to compare results of the different animal models concerning reliability, degree and

M AN U

387

SC

383

388

permanence of the induced lymphedema, it will be inevitable to define standardized parameters.

389

Experimental data have shown that phenotypic changes observed in lymphedema patients are present

390

and could define lymphedema in the experimental setting. As such are fibrosis, infiltration of immune

391

cells and deposition of adipose tissue.

392

the presence of lymphedema and the categorization in acute or chronic, based on the timeline in which

393

they appear. This further distinction into acute and chronic would enable a better approach of surgical

394

interventions attempted to tackle lymphedema. In addition, systemic changes in cytokines and growth

395

factors, associated with improved or impaired lymphatic function could be utilized to evaluate the

396

effect of the surgical intervention and compare the results with the clinical setting.40 Evaluation of

397

lymphatic function through in vivo imaging (microlymphography, lymphoscintigraphy, near-infrared

398

imaging) could offer more accurate and quantitative information for the standardization of

399

lymphedema.64, 65

401 402

EP

TE D

These could serve as the standardized parameters to define

AC C

400

54, 63

4.5 Lymphatic Surgery - Translational Issues Attempts to reduce experimental lymphedema reconstructing the lymphatic system date back

403

to the 1960ies. LVA22, 26 or lymphonodo-venous shunts21 were performed by several authors, mainly in

404

large animals. However, scarce results were reported. Baumeister et al66 introduced the transplantation

16

ACCEPTED MANUSCRIPT 405

of lymphatic vessels in dogs, showing significant reduction of early lymphedema. This method has

406

been applied in humans, resulting in complete symptomatic remission in 3 out of 14 patients.67

407

The treatment of secondary lymphedema with ALNT is a "hot topic" in clinical lymphatic surgery. In most countries, however, it has not been established and there are only few data and

409

anecdotal reports about the clinical results. Different authors have performed experimental ALNT30, 31,

410

36, 42, 50, 68

411

regenerate the lymphatic function most successfully. Honkonen et al31 justifiably pointed out that the

412

application of growth factors in a history of malignant disease is critical and the risk-benefit ratio is

413

hardly predictable. Yet, experimental data indicate that ALNT alone does not sufficiently restore the

414

lymphatic function and this combination might be of great value in treating secondary lymphedema.

415

Importantly, the experimental data produced are associated with short- or mid-term (<6 months)

416

follow-up, which does not give consideration to the lifelong course of human lymphedema. In

417

addition, the evaluation of the therapeutic approach is not consistent including often absence of

418

histological/immunohistochemical work-up of the specimens; thus, the impact of experimental ALNT

419

on subacute or chronic lymphedema remains unclear. Donor-side morbidity after ALNT has scarcely

420

been investigated in animal studies. Clinically, lymph node harvesting has been assessed with a

421

maximum follow-up of 56 months and did not show any relevant lymphatic dysfunction.69 Other

422

authors reported complication rates up to 38% following ALNT.70 In vascularized microsurgical

423

lymphatic tissue transfer the integration of the grafted lymph nodes may fail.71, 72 In summary, ALNT

424

is experimentally based on treating early lymphedema with short time follow-up.

EP

TE D

M AN U

SC

and the application of growth factors (VEGF-C) additional to ALNT has proven to

AC C

425

RI PT

408

The advances in the field of tissue engineering constitute a future challenge for lymphatic

426

surgery. Engineered lymphatic grafts propose a potential alternative to bridge defects involving

427

collecting lymphatic vessels or lymph nodes. Suematsu et al73 have produced immunologically active

428

engineered lymph nodes that, despite not promoting lymphatic function per se, create the foundation

429

for further advances. Engineering of collecting lymphatic vessels is still less advanced and restricted to

430

in vitro studies. The development of lymphatic grafts would absolve complications such as donor side

431

morbidity and could improve patients` recovery. The absence of robust preclinical study designs,

432

however, hampers the evaluation of such therapeutic interventions.

17

ACCEPTED MANUSCRIPT 433

It is worthy to underline that lymphatic surgery and its therapeutic translation is not only an

434

issue to technical skills but requires an integrative approach of lymphatic biology, mechanical

435

properties of the lymphatic vessels and engineering strategies in order to achieve solutions for the

436

treatment of lymphatic insufficiencies.

438

RI PT

437

5. CONCLUSIONS

439

None of the available lymphedema animal models to evaluate the effectiveness of surgical intervention is perfect. However, we should use the existing lymphedema animal models in a

441

complementary manner, since neither pure basic research on lymphedema nor the surgical approaches

442

have achieved a curative treatment yet. We understand that a reproducible and permanent

443

lymphedema model is required to further establish microsurgical lymphatic surgery. At present, the

444

rodent limb appears to be the most eligible and cost-effective model to investigate the reconstruction

445

of lymphatic function. Not least, it offers an easily available microsurgical training opportunity. Main

446

limitation is the small size of the animals, which enables spontaneous lymphedema resolution unless

447

excessive radiation is performed.

TE D

448

ACKNOWLEDGEMENT

450

EP

451

Special thanks go to Martina Gosteli, University of Zurich, for the systematic literature search and to Stefan Schwyter, University Hospital Zurich, for the animal drawing (Fig. 2).

452

AC C

449

453

FIGURE LEGEND

454

Fig. 1

457

Details of the study selection process. Modified from Moher et al, J Clin Epidemiol. 200917

455 456

M AN U

SC

440

Fig. 2

The spectrum of rodent lymphedema models

18

ACCEPTED MANUSCRIPT 458

REFERENCES

459

1.

460

6(3-4):109-22.

462

Angiogenesis 2014; 17(2):383-93. 3.

464 465

follow-up study and review. Pediatrics 1985; 76(2):206-18. 4.

466 467

Becker C, Arrive L, Saaristo A, et al. Surgical treatment of congenital lymphedema. Clin Plast Surg 2012; 39(4):377-84.

5.

468 469

Smeltzer DM, Stickler GB, Schirger A. Primary lymphedema in children and adolescents: a

SC

463

Aebischer D, Iolyeva M, Halin C. The inflammatory response of lymphatic endothelium.

RI PT

2.

Traboulsi EI, Al-Khayer K, Matsumoto M, et al. Lymphedema-distichiasis syndrome and

M AN U

461

Cueni LN, Detmar M. The lymphatic system in health and disease. Lymphat Res Biol 2008;

FOXC2 gene mutation. Am J Ophthalmol 2002; 134(4):592-6. 6.

Cormier JN, Askew RL, Mungovan KS, et al. Lymphedema beyond breast cancer: a systematic review and meta-analysis of cancer-related secondary lymphedema. Cancer 2010;

471

116(22):5138-49.

472

7.

TE D

470

McLaughlin SA, Wright MJ, Morris KT, et al. Prevalence of lymphedema in women with

473

breast cancer 5 years after sentinel lymph node biopsy or axillary dissection: Objective

474

measurements. J Clin Oncol 2008; 26(32):5213-5219.

476 477

Palliat Med 2005; 19(4):300-313. 9.

478

model of care. Cancer 2012; 118(8 Suppl):2237-49.

10.

481 482 483

Hayes SC, Johansson K, Stout NL, et al. Upper-body morbidity after breast cancer: incidence and evidence for evaluation, prevention, and management within a prospective surveillance

479 480

Williams AF, Franks PJ, Moffatt CJ. Lymphoedema: Estimating the size of the problem.

EP

8.

AC C

475

Goldsmith HS. Long term evaluation of omental transposition for chronic lymphedema. Ann Surg 1974; 180(6):847-9.

11.

Wallmichrath J, Baumeister R, Giunta RE, et al. [Update on special surgical approaches in the therapy for lymphedemas]. Handchir Mikrochir Plast Chir 2012; 44(6):334-42.

19

ACCEPTED MANUSCRIPT 484

12.

485 486

Becker C, Vasile JV, Levine JL, et al. Microlymphatic surgery for the treatment of iatrogenic lymphedema. Clin Plast Surg 2012; 39(4):385-98.

13.

Koshima I, Inagawa K, Urushibara K, et al. Supermicrosurgical lymphaticovenular anastomosis for the treatment of lymphedema in the upper extremities. J Reconstr Microsurg

488

2000; 16(6):437-42. 14.

490

combined with polyglycolic acid scaffolds: a pilot study. J Biotechnol 2010; 150(1):182-9. 15.

492 16.

494

and future. Lymphat Res Biol 2011; 9(3):159-67. 17.

496 497

18.

19.

20.

21.

22.

510

Das SK, Franklin JD, O'Brien BM, et al. A practical model of secondary lymphedema in dogs. Plast Reconstr Surg 1981; 68(3):422-8.

23.

508 509

Clodius L, Wirth W. A new experimental model for chronic lymphoedema of the extremities (with clinical considerations). Chirurgia Plastica 1974; 2(2):115-132.

506 507

Pflug JJ, Calnan JS. The experimental production of chronic lymphoedema. Br J Plast Surg 1971; 24(1):1-9.

504 505

Olszewski W, Machowski Z, Sokolowski J, et al. Experimental lymphedema in dogs. J Cardiovasc Surg (Torino) 1968; 9(2):178-83.

502 503

Danese CA, Georgalas-Bertakis M, Morales LE. A model of chronic postsurgical lymphedema in dogs' limbs. Surgery 1968; 64(4):814-20.

500 501

Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. J Clin Epidemiol 2009; 62(10):1006-12.

498 499

Mehrara BJ, Zampell JC, Suami H, et al. Surgical management of lymphedema: past, present,

M AN U

495

SC

development of artificial lymph nodes. Front Immunol 2012; 3:343.

TE D

493

Cupedo T, Stroock A, Coles M. Application of tissue engineering to the immune system:

EP

491

Dai T, Jiang Z, Li S, et al. Reconstruction of lymph vessel by lymphatic endothelial cells

AC C

489

RI PT

487

Chen HC, Pribaz JJ, O'Brien BM, et al. Creation of distal canine limb lymphedema. Plast Reconstr Surg 1989; 83(6):1022-6.

24.

Casley-Smith JR, Clodius L, Piller NB, et al. A model of lymphoedema in the rabbit's ear The effects of the benzopyrones. Chirurgia Plastica 1977; 4(1):5-14.

20

ACCEPTED MANUSCRIPT 25.

512

1983; 4(4):236-42. 26.

514

1990; 24(1):30-33.

516

recombinant VEGF-C. FASEB J 2002; 16(14):1985-7. 28.

518

following lymph node excision in sheep. J Vasc Res 2009; 46(5):426-34. 29.

520 521

lymphatics in sheep. Microvasc Res 2007; 73(3):214-23. 30.

522 523

31.

32.

33.

34.

Kanter MA, Slavin SA, Kaplan W. An experimental model for chronic lymphedema. Plast Reconstr Surg 1990; 85(4):573-80.

35.

532 533

Huang GK, Maekawa J. A new experimental model of lymphedema in rats'hind lower legs. Eur J of Plast Surg 1990; 13(5):192-197.

530 531

Wang GY, Zhong SZ. A model of experimental lymphedema in rats' limbs. Microsurgery 1985; 6(4):204-10.

528 529

Honkonen KM, Visuri MT, Tervala TV, et al. Lymph node transfer and perinodal lymphatic growth factor treatment for lymphedema. Ann Surg 2013; 257(5):961-7.

526 527

Lahteenvuo M, Honkonen K, Tervala T, et al. Growth factor therapy and autologous lymph node transfer in lymphedema. Circulation 2011; 123(6):613-20.

524 525

Jila A, Kim H, Nguyen VP, et al. Lymphangiogenesis following obstruction of large postnodal

SC

519

Tobbia D, Semple J, Baker A, et al. Lymphedema development and lymphatic function

M AN U

517

Szuba A, Skobe M, Karkkainen MJ, et al. Therapeutic lymphangiogenesis with human

RI PT

27.

TE D

515

Dominici C, De Leo S, Covarelli P, et al. Experimental model of lymphedema. Vasc Surg

EP

513

Huang GK, Hsin YP. An experimental model for lymphedema in rabbit ear. Microsurgery

AC C

511

Levine S, Saltzman A. Acute obstructive parasternal lymphedema produced in rats without surgery. Lymphology 1990; 23(4):187-93.

36.

Kawahira T, Sugimoto T, Okada M, et al. An experimental study of surgical treatment for

534

lymphedema in rats: A modified Kinmonth procedure and autologous lymph node capsule-

535

venous anastomosis with lymph node transfer. Ann Thorac Cardiovasc Surg 1999; 5(2):86-93.

536 537

37.

Kinmonth JB, Hurst PA, Edwards JM, et al. Relief of lymph obstruction by use of a bridge of mesentery and ileum. Br J Surg 1978; 65(12):829-33.

21

ACCEPTED MANUSCRIPT 38.

539

lymphedema. Lymphology 1999; 32(3):111-7. 39.

541 542

transfer for acute lymphedema. Ann Surg 1999; 229(3):421-7. 40.

543 544

Tabibiazar R, Cheung L, Han J, et al. Inflammatory manifestations of experimental lymphatic insufficiency. PLoS Med 2006; 3(7):e254.

41.

545 546

Slavin SA, Van den Abbeele AD, Losken A, et al. Return of lymphatic function after flap

RI PT

540

Lee-Donaldson L, Witte MH, Bernas M, et al. Refinement of a rodent model of peripheral

Karkkainen MJ, Saaristo A, Jussila L, et al. A model for gene therapy of human hereditary lymphedema. Proc Natl Acad Sci U S A 2001; 98(22):12677-82.

42.

Tammela T, Saaristo A, Holopainen T, et al. Therapeutic differentiation and maturation of

SC

538

lymphatic vessels after lymph node dissection and transplantation. Nat Med 2007;

548

13(12):1458-66. 43.

550

hind limb: a preliminary report. Ann Plast Surg 2012; 69(5):565-8. 44.

552

Experimental Reproduction in Animals. Ann Surg 1934; 100(4):812-32. 45.

554 555

46.

47.

al Assal F, Cordeiro AK, De Souza e Castro I. A new technique of microlympho-venous anastomoses. Experimental study. J Cardiovasc Surg 1988; 29(5):552-5.

48.

560 561

Puckett CL, Jacobs GR, Hurvitz JS, et al. Evaluation of lymphovenous anastomoses in obstructive lymphedema. Plast Reconstr Surgery 1980; 66(1):116-20.

558 559

Firica A, Ray A, Murat JE, et al. Alleviation of experimental lymphedema by lymphovenous anastomosis in dogs. Am Surg 1972; 38(7):409-12.

556 557

TE D

553

Homans J, Drinker CK, Field M. Elephantiasis and the Clinical Implications of Its

EP

551

Oashi K, Furukawa H, Oyama A, et al. A new model of acquired lymphedema in the mouse

AC C

549

M AN U

547

Kinjo O, Kusaba A. Lymphatic vessel-to-isolated-vein anastomosis for secondary lymphedema in a canine model. Surg Today 1995; 25(7):633-9.

49.

Baumeister RG, Seifert J, Wiebecke B, et al. Experimental basis and first application of

562

clinical lymph vessel transplantation of secondary lymphedema. World J Surg 1981; 5(3):401-

563

7.

564 565

50.

Chen HC, O'Brien BM, Rogers IW, et al. Lymph node transfer for the treatment of obstructive lymphoedema in the canine model. Br J Plast Surg 1990; 43(5):578-86.

22

ACCEPTED MANUSCRIPT 566

51.

567 568

Hadamitzky C, Pabst R. Acquired lymphedema: an urgent need for adequate animal models. Cancer Res 2008; 68(2):343-5.

52.

Tobbia D, Semple J, Baker A, et al. Experimental assessment of autologous lymph node transplantation as treatment of postsurgical lymphedema. Plast Reconstr Surg 2009;

570

124(3):777-86.

571

53.

572 573

RI PT

569

Cheung L, Han J, Beilhack A, et al. An experimental model for the study of lymphedema and its response to therapeutic lymphangiogenesis. BioDrugs 2006; 20(6):363-70.

54.

Rutkowski JM, Moya M, Johannes J, et al. Secondary lymphedema in the mouse tail:

Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9. Microvasc

575

Res 2006; 72(3):161-71.

577

the mouse tail skin. Am J Physiol Heart Circ Physiol 2008; 294(3):H1326-34. 56.

579

10 expression in mice with lymphatic dysfunction. J Leukoc Biol 2013; 94(1):137-45. 57.

581 582

regeneration during wound repair. Am J Physiol Heart Circ Physiol 2008; 295(5):H2113-27. 58.

583 584

59.

Shesol BF, Nakashima R, Alavi A, et al. Successful lymph node transplantation in rats, with restoration of lymphatic function. Plast Reconstr Surg 1979; 63(6):817-23.

60.

587 588

Sommer T, Buettner M, Bruns F, et al. Improved regeneration of autologous transplanted lymph node fragments by VEGF-C treatment. Anat Rec (Hoboken) 2012; 295(5):786-91.

585 586

Clavin NW, Avraham T, Fernandez J, et al. TGF-beta1 is a negative regulator of lymphatic

TE D

580

Kimura T, Sugaya M, Blauvelt A, et al. Delayed wound healing due to increased interleukin-

EP

578

Uzarski J, Drelles MB, Gibbs SE, et al. The resolution of lymphedema by interstitial flow in

M AN U

55.

AC C

576

SC

574

Can J, Cai R, Li S, et al. Experimental study of lymph node auto-transplantation in rats. Chin Med J (Engl) 1998; 111(3):239-41.

61.

Kriederman BM, Myloyde TL, Witte MH, et al. FOXC2 haploinsufficient mice are a model

589

for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet 2003;

590

12(10):1179-85.

591

62.

Makinen T, Jussila L, Veikkola T, et al. Inhibition of lymphangiogenesis with resulting

592

lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat Med 2001;

593

7(2):199-205.

23

ACCEPTED MANUSCRIPT 594

63.

Zampell JC, Aschen S, Weitman ES, et al. Regulation of adipogenesis by lymphatic fluid

595

stasis: part I. Adipogenesis, fibrosis, and inflammation. Plast Reconstr Surg 2012; 129(4):825-

596

34. 64.

598 65.

600 601

66.

Baumeister RG, Seifert J. Microsurgical lymphvessel-transplantation for the treatment of lymphedema: experimental and first clinical experiences. Lymphology 1981; 14(2):90.

67.

604 605

Blum KS, Proulx ST, Luciani P, et al. Dynamics of lymphatic regeneration and flow patterns after lymph node dissection. Breast Cancer Res Treat 2013; 139(1):81-6.

602 603

RI PT

2012; 22(1):12-3.

SC

599

Proulx ST, Detmar M. Watching lymphatic vessels grow by making them glow. Cell Res

Felmerer G, Sattler T, Lohrmann C, et al. Treatment of various secondary lymphedemas by microsurgical lymph vessel transplantation. Microsurgery 2012; 32(3):171-7.

68.

M AN U

597

Fu K, Izquierdo R, Vandevender D, et al. Transplantation of lymph node fragments in a rabbit

606

ear lymphedema model: a new method for restoring the lymphatic pathway. Plast Reconstr

607

Surg 1998; 101(1):134-41.

609

microvascular lymph node transfer. Plast Reconstr Surg 2012; 130(6):1246-53. 70.

611 612

transplantation for limb lymphoedema. Eur J Vasc Endovasc Surg 2013; 45(5):516-20. 71.

613 614

617 618

Becker C, Assouad J, Riquet M, et al. Postmastectomy lymphedema: long-term results following microsurgical lymph node transplantation. Ann Surg 2006; 243(3):313-5.

72.

615 616

Vignes S, Blanchard M, Yannoutsos A, et al. Complications of autologous lymph-node

EP

610

Viitanen TP, Maki MT, Seppanen MP, et al. Donor-site lymphatic function after

TE D

69.

AC C

608

Saaristo AM, Niemi TS, Viitanen TP, et al. Microvascular breast reconstruction and lymph node transfer for postmastectomy lymphedema patients. Ann Surg 2012; 255(3):468-73.

73.

Suematsu S, Watanabe T. Generation of a synthetic lymphoid tissue-like organoid in mice. Nat Biotechnol 2004; 22(12):1539-45.

ACCEPTED MANUSCRIPT

Table 1 - The Systematic Review Eligibility

Studies on animal models in lymphedema research.

Exclusion

Clinical studies, reviews and studies with experimental animal models not concerning lymphedema.

RI PT

The search was conducted on October 30st 2013 in six libraries (Medline,

Literature search

EMBASE, Scopus, Web of Science, Biosis and Cochrane) without time or language restriction.

Two independent reviewers (FSF/NL) included relevant studies by title and

Study selection

SC

abstract. The full text of potentially eligible studies was retrieved and

independently assessed for eligibility. Disagreement under the reviewers was

M AN U

resolved through discussion with an additional reviewer (FR). Studies describing new or partially new models (key articles) were included in

Data collection

chronological order for detailed analysis and data extraction. A narrative synthesis of each key article was provided. Parameters for data extraction were the following:

TE D

1) Animal/number of animals

2) Method to induce lymphedema 3) Quantification of lymphedema 4) Therapeutic approaches

EP

5) Reliability of producing lymphedema/Pitfalls of surgical procedure 6) Maximum follow-up

AC C

7) Accessibility for surgical treatment, required microsurgical skills and equipment (see Table 2)

Two reviewers independently performed the assessment of parameters; disagreement was resolved as mentioned above.

Outcomes of interest Primary

Assessment of existing animal models in lymphedema research

Secondary

To compare the different models and analyze the accessibility for surgical treatment/the required microsurgical skills

ACCEPTED MANUSCRIPT

Table 2 - Classification of Surgical Skills/Equipment ⇑

Basic surgical and microsurgical skills, loop magnification.

⇑⇑

Advanced surgical procedure, microsurgery

⇑⇑⇑

RI PT

(dissection). Microscope advantageous. Advanced microsurgical skills required (anastomosis, flap design/harvesting). Microscope required. ⇓

AC C

EP

TE D

M AN U

SC

No surgery required

ACCEPTED MANUSCRIPT

Table 3 - Canine Models Model N

LEA Induction

LE Quantification

Treatment

Danese et al18 (1968)

Hind limb

20

Surgery

Lymphangiography Histology

Ø

Olszewski et al19 (1968)

Hind limb

23

Surgery

Pflug et al20* (1971)

Hind limb

7

Obstruction

Clodius et al21* (1974)

Hind limb

5 10

Total lymph block + Etheron Deep lymph block + Etheron

Das et al22* (1981)

Hind limb

8 15

Surgery/Radiation Radiation/Surgery

Chen et al23* (1989)

Hind limb

17 10

Surgery/Radiation Surgery

Lymphovenous anastomosis

C

Required surgical skills/equipment (see Table 2)

* No histology

Ø

Max. Follow-Up Surgeon's (Months) FocusC

Mortality rate 5/20 LE production in 75%

>12

Permanent LE in 35% Follow-up > 1 year

>12

SC

M AN U

TE D

B

Limb circumference Lymphoscintigraphy

EP

Lymphedema

Limb circumference Lymphography Ø Histology Clinical swelling Lymphography Ø Venous flow Lymph/Tissue Protein Content LVAB/ Clinical swelling Free omentum Lymphography transpl. Limb circumference Lymphography LVA Water displacement volumetry

AC C

A

Reliability, Pitfalls

RI PT

Author (Year)

⇑

⇑

LE in 4/7 dogs after 4 months

5

⇓

Total lymph block: Lethal protein loss

3 weeks 5 months

⇑

Mortality rate 7/23 S/R: Chronic LE in 2/6 R/S: Chronic LE in 10/10

14

⇑

S/R with reliable chronic LE induction (12/14)

12

⇑

ACCEPTED MANUSCRIPT

Table 4 - Rabbit, Sheep & Pig Models Model

N

LEA Induction

LE Quantification

Treatment

Casley-Smith et al24 (1977)

Rabbit Ear

11

Surgery

Clinical swelling Electron microscopy

Benzopyrones

Huang et al25 (1983)

Rabbit Ear

50

Surgery

Ear thickness Ear Volume Ø Skin Thickness Lymphatic diameter/Histology

Dominici et al26* (1990)

Rabbit Ear

xa

Surgery Electrocoagulation

Water displacement volumetry LVAC Ear thickness

Szuba et al27 (2002)

Rabbit Ear

18

Surgery

Tobbia et al28 (2009)

Sheep 41 Hind limb

Surgery

Lahteenvuo et al30 Pig 14 (2011) Hind limb

Surgery

Surgery

A

Lymphedema

High success rate (47/50)

4-6

⇑⇑

Local anesthesia (No animal loss) Acute LE

30 days

⇑⇑

VEGF-CD

Success rate 16/18

2

⇑⇑

Ø

Success rate 100% LE declination over time

4

⇑

ALNTF + VEGF-C ALNT + VEGF-DG

Model with acute lymphatic damage, no LE

2

⇑

ALNT + intranodal VEGF-C ALNT + perinodal VEGF-C

Model with acute lymphatic damage, no LE

2

⇑

SC

8

M AN U

TE D

Seroma amount Lymphangiography Immunohistochemistry

EP

Pig 23 Hind limb

Water displacement volumetry Lymphoscintigraphy Immunohistochemistry Limb circumference Fluoroscopy Immunohistochemistry Protein transport (125I-HSAE) Seroma amount Lymphangiography Immunohistochemistry

⇑⇑

E

Radiolabeled human serum albumin

F

Autologous lymph node transplantation (mimicked by preservation of vascular lymph node pedicle)

B

Required surgical skills/equipment (see Table 2)

C

Lymphovenous anastomosis

G

Vascular endothelial growth factor D

D

Vascular endothelial growth factor C

a

No number reported

* No histology/immunohistochemistry

Max. Follow-Up Surgeon's FocusB (Months)

Benzopyrones reduce tissue edema/excess protein

AC C

Honkonen et al31 (2013)

Reliability/Pitfalls

RI PT

Author (Year)

ACCEPTED MANUSCRIPT

Table 5 - Rodent Models N

LEA Induction

Wang et al32 (1985)

Rat Hind limb

70

Surgery

Huang et al33 (1990)

Rat Hind limb

50

Surgery

Kanter et al34 (1990)**

Rat Hind limb

28

Surgery & Radiation

Levine et al35 (1990)

Rat Parasternal

220

Obstruction Histopathology

Kawahira et al36 (1999)

Rat Hind limb

26

Surgery

Lee-Donaldson et al38 Rat (1999)** Hind limb

45

Surgery & Radiation

Slavin et al39 (1999)**

23 29

Surgery Cautery

Mouse Tail

Karkkainen et al41 (2001)

Mouse (primary LE)

9

⇑⇑

High success rate (100%)

6.5

⇑⇑

Mortality rate 8% Stable LE production

9

⇑⇑

Ø

Only acute LE inducible

7 days

⇓

Kinmonth procedure ALNTB

LE occurred after 3 weeks

14 days*

⇑⇑⇑

Ø

Stable LE for up to 100 days (S/R and R/S)

100 days

⇑⇑

Pedicled rectus abdominis flap (myocutaneous)

Rat tail difficult to evaluate for LE (low tissue compliance)

3.5*

⇑⇑

Ø

Reliable LE induction Short follow-up (acute LE)

14 days

⇑⇑

Virus-mediated VEGF-CG gene therapy (intradermal)

Possible systemic effect of VEGF-C (i.e. tumor lymphangiogenesis)

-

⇓

Ø

TE D

Limb circumference Histopathology Limb volume Lymphangioscintigraphy MRIC Refractometry Tail diameter Lymphoscintigraphy FMD

45

Tail volume Lymphoscintigraphy Histology/IHCE Immune traffic imaging Arterial perfusion Cutaneous gene expression Fluorescence microscopy MRI FM IHC

Cautery

VEGFR-3F Mutation

Max. Follow-Up Surgeon's FocusH (Months)

High mortality rate (21%)

Ø

Ø

M AN U

Limb circumference Lymphoscintigraphy

Reliability/Pitfalls

EP

Tabibiazar et al40 (2006)

Treatment

Limb circumference Limb volume Histopathology Limb circumference Lymphatic diameter/contraction Histopathology

AC C

Rat, tail Mouse, tail

LE Quantification

RI PT

Model

SC

Author (Year)

ACCEPTED MANUSCRIPT

Tammela et al42 (2007)

Mouse Upper limb

Groups of 3

Surgery

Microlymphangiography Lymph node allograft MRI Growth factor treatment IHC, Immunocytochemistry (VEGF-C, VEGF-D)

Growth factor application can induce functional lymphatic vessels

6*

⇑⇑⇑

Oashi et al43 (2012)

Mouse Hind limb

20

Surgery & Radiation

Limb volume ICGI fluorescence IHC

Dissection of lymphatics and lymph nodes (subiliacal/popliteal)

6

⇑⇑⇑

RI PT

Ø

Lymphedema

E

Immunohistochemistry

* Follow-Up after LE treatment

B

Autologous lymph node transfer (lymph node

F

Vascular endothelial growth factor 3

** No histology/immunohistochemistry

capsulo-venous anastomosis)

G

Vascular endothelial growth factor C

C

Magnetic resonance imaging

H

Required surgical skills/equipment (see Table 2)

D

Fluorescent microlymphangiography

I

AC C

EP

M AN U

TE D

Indocyanine green

SC

A

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT