# Local heating-induced cutaneous vasodilation in reinnervated and noninnervated deep inferior epigastric perforator flaps

Hansje Smeele, Lisa Martin, Sander van Kuijk, Pieter Zwanenburg, René van der Hulst, Stefania Tuinder, Bérengère Fromy

► To cite this version:

Hansje Smeele, Lisa Martin, Sander van Kuijk, Pieter Zwanenburg, René van der Hulst, et al., Local heating-induced cutaneous vasodilation in reinnervated and noninnervated deep inferior epigastric perforator flaps. Microsurgery, 2023, 44 (1), pp.e31122. 10.1002/micr.31122. hal-04749510

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Local heating-induced cutaneous vasodilation in reinnervated and noninnervated deep inferior epigastric perforator flaps

Hansje P. Smeele, MD¹; Lisa Martin, MSc²; Sander M. J. van Kuijk, PhD³; Pieter R. Zwanenburg, MD⁴; René R. W. J. van der Hulst, MD PhD¹; Stefania M. H. Tuinder, MD PhD¹*; Bérengère Fromy, PhD²*

1. Hansje P. Smeele, MD; René R. W. J. van der Hulst, MD PhD; Stefania M. H. Tuinder, MD PhD. Department of Plastic, Reconstructive and Hand Surgery, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Center; Maastricht, the Netherlands.

2. Lisa Martin, MSc; Bérengère Fromy, PhD. Functional Integrity of the Skin Group, Tissue Biology and Therapeutic Engineering Laboratory (LBTI), CNRS UMR5305, Université Lyon 1; Lyon, France.

3. Sander M. J. van Kuijk, PhD. Department of Clinical Epidemiology and Medical Technology Assessment, Maastricht University Medical Center; Maastricht, the Netherlands.

4. Pieter R. Zwanenburg, MD. Department of Surgery, Amsterdam Gastroenterology and Metabolism, Amsterdam Infection & Immunity, Amsterdam UMC, University of Amsterdam; Amsterdam, the Netherlands.

* Authors have made an equal contribution to the work and are co-last authors.

**Corresponding author**

Bérengère Fromy, PhD
Functional Integrity of the Skin Group, Tissue Biology and Therapeutic Engineering
Laboratory (LBTI)
CNRS UMR5305
Université Lyon 1
7 Passage du Vercors
69367 Lyon, France
berengere.fromy@univ-lyon1.fr
+33(0)472722686

**Funding statement**

This study was financially supported by the Van Gogh Programme, which granted an exchange scholarship (VGP.20/00034) for joint data collection. The funding organization was not involved in design and conduct of the study.
**Conflict of interest disclosure**

The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.

**Word count**

- Abstract: 323

- Manuscript (Introduction, Methods, Results, Discussion): 3,070

**Tables and figures**

- Tables: 4

- Figures: 4
1 **ABSTRACT (323 words)**

2 **Introduction** Cutaneous vascular reactivity to local heating in free flaps has not been
3 characterized. We aimed to assess local heating-induced cutaneous vasodilation in
4 reinnervated and noninnervated deep inferior epigastric perforator (DIEP) flaps.

5 **Methods** We conducted a cross-sectional study of 21 female patients with an
6 uncomplicated unilateral delayed DIEP breast reconstruction at least two years after
7 surgery. DIEP flaps and contralateral breasts were subjected to direct local heating, and
8 skin blood flow was assessed using laser-Doppler flowmetry. To evaluate sensory-nerve-
9 fiber function, touch perception thresholds were assessed using a 20-piece Touch-test™
10 Sensory Evaluator, and cutaneous warm detection and heat pain thresholds were
11 measured using a TSA-II device.

12 **Results** Of the participants, 10 had a reinnervated DIEP flap with a single coapted nerve
13 (mean flap weight, 610 ± 296 g) and 11 had a noninnervated DIEP flap (mean flap
14 weight, 613 ± 169 g). Mean age was 58 ± 11 years, mean follow-up time was 5 ± 1
15 years, and mean BMI was 24 ± 3 kg/m². DIEP flaps exhibited significantly weaker
16 cutaneous vasodilation in response to local heating than contralateral breasts (median
17 peak skin blood flow, 59 [25ᵗʰ–75ᵗʰ percentile, 36 – 71] a.u. for DIEP flaps versus 94 [74
18 – 141] a.u. for contralateral breasts; *p* < 0.001). The magnitude of the response was
19 similar between reinnervated and noninnervated flaps (median peak skin blood flow, 55
20 [25ᵗʰ–75ᵗʰ percentile, 39 – 68] a.u. for reinnervated DIEP flaps versus 66 [36 – 77] a.u.
21 for noninnervated DIEP flaps; *p* = 0.75). Of participants with reinnervated DIEP flaps,
22 90% perceived heat pain below the 50°C safety threshold, as compared to 36% of
23 participants with noninnervated DIEP flaps (two-tailed *p* = 0.02).

24 **Conclusion** Our results suggest that free flap transfer causes longstanding impairment,
25 yet not complete abolition, of both the sensory nerve-mediated and nitric oxide-
26 dependent local heating-induced cutaneous vasodilatory systems. We found no statistical
27 evidence that flap reinnervation improves the ability to raise skin blood flow in response
28 to local heating.
29 **Keywords:** local heating; skin blood flow; vasodilation; breast reconstruction; nerve

30 coaptation
31 **INTRODUCTION (358 words)**

32 The deep inferior epigastric perforator (DIEP) flap consists of skin and subcutaneous
33 tissue harvested from the lower abdomen along with its blood supply. The DIEP flap is
34 ideally suited for breast reconstruction after mastectomy, yielding high levels of patient-
35 reported aesthetic outcomes.¹,² Nevertheless, sensory outcomes are often poor due to
36 nerve disruption with flap harvest. Patients mourn reduced or absent sensation in their
37 reconstructed breast whilst dressing, taking a shower, and in intimate settings.³
38 Protective sensation too is often lost, as is illustrated by reports of burns to breast
39 reconstruction flaps induced by heat exposure (e.g., sun, shower, and hot water bottles)
40 up to five years after surgery.⁴⁻⁶ These burns might ensue from lost ability to register
41 heat, but sparing of adjacent skin indicate that the flap struggles to actively protect itself
42 against heat, too.

43 Healthy human skin eliminates heat through vasodilation. Whole body passive heating
44 elevates core temperature, which evokes an increase in skin blood flow mediated
45 predominantly by sympathetic cholinergic nerves.⁷ Klasson and colleagues observed
46 normal cutaneous vasodilation in response to whole body heating in DIEP flaps at a
47 median of 1.3 years after surgery.⁸ Direct local heating of hairy skin elicits cutaneous
48 vasodilation in the heated area only, a response largely dependent on sensory nerve
49 endings and nitric oxide.⁹⁻¹¹ Whether DIEP flaps (or indeed any free flap) exhibit an
50 adequate cutaneous vasodilator response to local heating has not been examined.

51 Surgical coaptation of a sensory nerve supplying the DIEP flap to a sensory nerve at the
52 mastectomy site is thought to help restore sensory supply by guiding axonal
53 regeneration, a premise supported by enhanced sensory detection ability observed in
54 reinnervated flaps.¹² We hypothesized that as part of the cutaneous vasodilator response
55 to local heating is sensory nerve-mediated, the response would be superior in
56 reinnervated flaps, where neural continuity has been partially restored.
The study aims were to examine cutaneous vasodilation in response to local heating in DIEP flaps, and to evaluate whether flap reinnervation might enhance the response. We also explored which factors might explain variation in the magnitude of the response. We compared vasodilation results against sensory detection thresholds to infer cutaneous nerve fiber function.
63 **Design**

64 We performed a cross-sectional study. Study participants were recruited between April
65 2021 and December 2021. Experiments were conducted in July 2021 and January 2022
66 at Maastricht University Medical Center; Maastricht, the Netherlands.

67 **Participants**

68 The protocol was approved by the institutional ethics committee. Participants gave
69 informed consent.¹³ We included female patients with an uncomplicated unilateral
70 delayed DIEP breast reconstruction at least two years after surgery to allow peripheral
71 nerves within the flap time to regenerate.¹⁴ Exclusion criteria were a history of cancer in
72 the contralateral breast, absence of a DIEP skin island, and conditions known to impair
73 peripheral nerve regeneration (i.e., post-reconstruction radiotherapy¹⁵, active smoking¹⁶,
74 and diabetes mellitus¹⁷). Peripheral neuropathy, prevalent in the study population, was
75 not an exclusion criterium but a covariable to ensure a large enough pool of eligible
76 patients.

77 **Surgical technique**

78 Unilateral, delayed DIEP flap breast reconstructions were performed according to the
79 conventional technique.¹⁸ DIEP flaps were reinnervated with a single coapted nerve upon
80 patient request. A sensory branch of the 10th to 12th intercostal nerve was dissected as
81 a donor nerve, and the anterior cutaneous branch of the second or third intercostal nerve
82 as recipient nerve.¹⁹ After completing vascular anastomoses and flap inset, direct end-to-
83 end coaptation was performed using epineural sutures and fibrin glue. No nerve conduits
84 or nerve grafts were used.

85 **Variables**

86 In healthy humans, local heating of hairy skin induces a biphasic response.¹⁰ Our primary
87 outcome was the rapid increase in skin blood flow within the first 3 to 5 minutes of local
88 heating, a reaction that relies predominantly on local activity of sensory nerve endings.
Our secondary outcome was the slower vasodilation phase that attains a plateau in response to sustained local heating, a reaction mediated mainly by nitric oxide.⁹,¹⁰

Additional outcomes were cutaneous mechanical detection, warm detection, and heat pain thresholds as surrogates of sensory nerve function. Owing to combinations of factors including patient preference, technical feasibility, and surgeon perseverance, not all DIEP flaps were reinnervated at the time of reconstruction. The independent variable of interest was measurement site (i.e., contralateral breasts, reinnervated DIEP flaps, and noninnervated DIEP flaps). Observers were blinded to DIEP innervation status; participants were not. Data extracted from medical records comprised age, body mass index, menopausal status, history of smoking, peripheral neuropathy, hypertension, hypercholesterolemia, anti-cancer treatments, flap type, flap weight, ischemia time, follow-up time (i.e., the interval between breast reconstruction and experiment), and contralateral breast symmetrization.

## Local heating-induced cutaneous vasodilatation

Skin blood flow in response to direct local heating was monitored using laser-Doppler flowmetry (PeriFlux System 6000; Perimed, Stockholm, Sweden). We tested participants' DIEP flaps and contralateral breasts (figure 1). One site per flap or breast was tested; scars were avoided. After 10 minutes of supine acclimatization in a quiet, constant-temperature room, data collection started with a one-minute normothermic baseline followed by 20 minutes of local heating at 42°C (457-PI probe; Perimed, Stockholm, Sweden). Skin temperature was measured before onset of local heating; control skin blood flow was continuously recorded 3 to 5 cm away from the heated area (440 probe; Perimed, Stockholm, Sweden). Blood flow recordings were analyzed in arbitrary units (a.u.). Before the protocol, core temperature was measured with an infrared ear thermometer (ThermoScan Pro 6000; Braun, Melurgeon, Germany). Systolic and diastolic blood pressures were measured using a Gamma XXL LF sphygmomanometer (Heine, Germany) directly before and after the protocol.

## Cutaneous quantitative sensory testing
Touch perception thresholds were assessed using a 20-piece Touch-test™ Sensory Evaluator (North Coast Medical, Inc., Gilroy, CA, USA). Monofilaments were applied three times in an ascending fashion.<sup>14,20</sup> An affirmative response was recorded when participants felt the stimulus at least one out of three times and could accurately locate it. Scores were converted into target stress (g/mm<sup>2</sup>). Cutaneous warm detection and heat pain thresholds were measured using a TSA-II device (Medoc, Ramat-Yishai, Israel) with a 9.0 cm<sup>2</sup> Peltier-type thermode. Participants were asked to abort a thermal stimulus increasing from 32°C to 50°C at a rate of 1°C/s when perceived. Warm detection and heat pain thresholds were both tested three times; for each series, the mean was recorded as the final value. If a warm detection or heat pain threshold was not reached within the safety limit, 50°C was entered for analysis.

## Nerve fiber integrity

We histologically examined dermal and epidermal small sensory nerve fibers in a DIEP skin island excised as a secondary procedure. The tissue originated from a 39-year-old patient who had unilateral breast cancer. She was treated with neoadjuvant chemotherapy, trastuzumab, mastectomy, and tamoxifen. Sixteen months before skin island excision, she had an uncomplicated delayed bipedicled DIEP breast reconstruction with dual nerve coaptation. The DIEP flap weighted 895 grams. The patient never smoked and was healthy apart from breast cancer. Mechanical detection, warm detection, and heat pain thresholds were obtained before surgery; skin blood flow in response to local heating could not be assessed because the laser-Doppler device was unavailable. Immediately after excision, the skin island was fixed in 4% buffered formaldehyde solution and stored at 4°C for 24 hours. After rinsing in PBS RNAse free solution, the sample was immersed in 10% sucrose for 24 hours at 4°C. The next day, the tissue was immersed in isopentane for ~30-60 seconds and stored at -80°C. Sections 30 µm thick were prepared using Leica CM3050 S Cryostat and stored at -80°C. After two hours of curing at room temperature, sections were saturated with 5% goat serum, 2% BSA, and 0.1% Tween-20 for one hour. Sections were incubated overnight in a
145 humidified chamber at 4°C with primary rabbit antibody (1:100) against human protein
146 gene product 9.5 (PGP9.5; ab27053), to identify dermal and epidermal neuronal
147 structures. They were then washed with 1X-PBS and incubated for 45 minutes with goat
148 anti-rabbit Alexa-546 (1:1000; A-11035) at room temperature in a dark chamber. After
149 washing, sections were mounted with Fluoroshield™ with DAPI medium (F6057) to
150 visualize cell nuclei, and examined using a Nikon Ti-E Fluorescence Motorized microscope
151 under 40× magnification.

152 **Data analysis**

153 Participants and DIEP flaps were characterized using counts and percentages for
154 categorical variables, means and standard deviations for normally distributed variables
155 (Shapiro-Wilk *p* ≥ 0.01), and medians and 25th and 75th percentiles for other variables.
156 The paired-samples Wilcoxon Signed Rank Test was used for comparisons between
157 contralateral breasts and DIEP flaps. Normally distributed data were compared between
158 reinnervated and noninnervated flaps using the independent samples t-test, and other
159 data using the Mann-Whitney *U* test. Data were compared between contralateral breasts,
160 reinnervated DIEP flaps, and noninnervated DIEP flaps using one-way analysis of
161 variance for dependent samples followed by Tukey's multiple-comparison test. Factors
162 that might explain variation in peak and plateau skin blood flow were identified using
163 univariable linear regression analyses. Spearman correlation coefficients (*r*<sub>s</sub>) were used to
164 estimate associations between variables. All statistical analyses were performed using R
165 version 4.0.5 (R Foundation for Statistical Computing, Vienna, Austria), with *α* = 0.05
166 unless otherwise stated.
167 **RESULTS (518 words)**

168 **Participants**

169 Of 661 patients who underwent a DIEP breast reconstruction between January 2010 and
170 July 2019, 101 were eligible for inclusion, 51 of whom had a reinnervated flap. Twenty-
171 one patients agreed to participate, 10 of whom had a reinnervated flap (mean flap
172 weight, 610 ± 296 g) and 11 of whom had a noninnervated flap (mean flap weight, 613
173 ± 169 g). Mean age was 58 ± 11 years, mean follow-up time was 5 ± 1 years, and mean
174 BMI was 24 ± 3 kg/m². No statistically significant differences in baseline characteristics
175 between outcome groups were observed (table 1). All but one participant were
176 postmenopausal; none took hormone replacement therapy. Four patients with
177 reinnervated flaps had contralateral breast symmetrization (40%) as compared to two
178 patients with noninnervated flaps (18%). Data completeness was 100%.

179 **Local heating-induced cutaneous vasodilation**

180 In both contralateral breasts and DIEP flaps, local heating to 42°C evoked a biphasic
181 increase in skin blood flow, characterized by an initial peak followed by slower
182 vasodilatation attaining a plateau (figure 2A-C). DIEP flaps exhibited weaker cutaneous
183 vasodilation in response to local heating than contralateral breasts (median peak skin
184 blood flow, 59 [25ᵗʰ–75ᵗʰ percentile, 36 – 71] a.u. for DIEP flaps *versus* 94 [74 – 141]
185 a.u. for contralateral breasts; *p* < 0.001; table 2). Areas under the curve were
186 significantly greater in contralateral breasts than in DIEP flaps (figure 2D). Peak and
187 plateau skin blood flow did not differ between reinnervated and noninnervated DIEP flaps
188 (median peak skin blood flow, 55 [25ᵗʰ–75ᵗʰ percentile, 39 – 68] a.u. for reinnervated
189 DIEP flaps *versus* 66 [36 – 77] a.u. for noninnervated DIEP flaps; *p* = 0.75). Univariable
190 linear regression analysis showed that chemotherapy was inversely associated with peak
191 and plateau skin blood flow in both contralateral breasts and DIEP flaps (table 3).
192 **Cutaneous quantitative sensory testing**

193 Compared with contralateral breasts, cutaneous mechanical detection, warm detection,
194 and heat pain thresholds were elevated in reinnervated DIEP flaps and further elevated in
195 noninnervated DIEP flaps (figure 3). Significantly more participants with reinnervated
196 DIEP flaps felt heat pain below the 50°C safety limit (table 4). As heat pain threshold and
197 peak skin blood flow both rely on functional small unmyelinated heat-sensitive C-fibers<sup>21</sup>,
198 we expected an inverse correlation between these variables; however, this was neither
199 the case in contralateral breasts (r<sub>s</sub> = 0.11, 95% CI = -0.38 - -0.55; p = 0.63) nor in
200 DIEP flaps (r<sub>s</sub> = 0.17, 95% CI = -0.22 - -0.54; p = 0.46). Mechanical detection threshold
201 and peak skin blood flow were uncorrelated in contralateral breasts (r<sub>s</sub> = 0.04, 95% CI =
202 -0.41 - -0.49; p = 0.88) and in DIEP flaps (r<sub>s</sub> = 0.20, 95% CI = -0.27 - -0.60; p = 0.39).

203 **Nerve fiber integrity**

204 The mechanical detection threshold of the examined reinnervated skin island was 68.2
g/mm²; neither warmth nor heat were perceived below the 50°C safety limit. In healthy
205 abdominal control skin of a 40-year-old woman, we observed small sensory nerve fibers
206 mainly in the dermis, but also in the epidermis (figure 4A). Conversely, the reinnervated
207 DIEP skin island showed a complete loss of dermal and epidermal small sensory nerve
208 fibers (figure 4B).
209
210 DISCUSSION (969 words)

211 DIEP flaps three to seven years after surgery exhibited classic biphasic cutaneous vasodilation in response to local heating. However, peak and plateau skin blood flow in DIEP flaps were significantly lower than in contralateral breasts. These findings indicate that free flap transfer causes longstanding impairment, yet not complete abolition, of both the sensory nerve-mediated and nitric oxide-dependent local heating-induced cutaneous vasodilatory systems. This has not been demonstrated before.

217 In healthy human skin, small unmyelinated heat-sensitive C-fibers respond to local heating by releasing neurotransmitters that cause a rapid increase in skin blood flow.¹⁰,²¹ Above a certain threshold, the same C-fibers transmit an action potential to the brain, resulting in conscious heat pain perception. As such, the observed attenuated peak skin blood flow in response to local heating (figure 2B-C) and elevated heat pain threshold (figure 3C) both provide evidence for C-fiber dysfunction in DIEP flaps. Elevated mechanical and warm detection thresholds (figure 3A and 3B) indicate additional dysfunction of large myelinated Aβ-fibers and small thinly myelinated Aδ-fibers, respectively.²² These findings resonate with clinical manifestations of sensory dysfunction in DIEP flaps described in the introduction.

227 Nine participants (43%) had a history of small fiber neuropathy. This condition damages peripheral autonomic and somatic small sensory nerve fibers, and has been associated with diminished sensory nerve-mediated cutaneous vasodilation in response to local heating.²³ Absence of an inverse association between small fiber neuropathy and peak skin blood flow in our sample might be due to heterogeneous neuropathy etiology and duration.

233 Not only peak but also plateau skin blood flow in response to local heating was significantly weaker in DIEP flaps than in contralateral breasts. This latter phase is primarily (though not exclusively) mediated by nitric oxide, meaning that free flap transfer might alter nitric oxide production, vascular responsiveness to nitric oxide, or
237 both. Although still under debate, the plateau vasodilator phase probably requires intact
238 sympathetic adrenergic function for its full expression.¹¹ The constrained plateau phase in
239 DIEP flaps could therefore be a symptom of cutaneous sympathetic denervation.

240 Prevailing theory indeed attributes pathological burns in breast reconstruction flaps to
241 sympathetic dysfunction.⁴⁻⁶ Contrary to this theory, Klasson and colleagues found normal
242 cutaneous sympathetic nerve-mediated vasodilation in response to whole body heating in
243 DIEP flaps.⁸ Also, our experiments were conducted at unheated skin temperatures where
244 adrenergic vasoconstrictor activity maintains resting cutaneous vascular tone (~32°C).

245 Unaltered baseline skin blood flow in DIEP flaps (table 2) refutes attenuated
246 vasoconstrictor tone and, by extension, sympathetic denervation.¹⁰,²⁴ Which exact
247 mechanisms impair the cutaneous plateau vasodilator response to local heating in DIEP
248 flaps could be clarified by studying effects of pharmacologic inhibitors.

249 Significantly more participants with reinnervated DIEP flaps were able to feel heat pain
250 below the 50°C safety limit than participants with noninnervated flaps (90% versus 36%;
251 table 4). The fact that the histologically examined tissue showed no signs of C-fiber
252 regeneration (i.e., no small sensory nerve fibers identified upon histological examination
253 [figure 4B] and no heat pain perception below 50°C) despite dual nerve coaptation might
254 lie with the relatively short follow-up time of 1.3 years.

255 Various confounding factors may have influenced the magnitude of the vasodilator
256 response to local heating. Exploratory analyses showed an inverse association between
257 chemotherapy and peak and plateau skin blood flow in contralateral breasts and DIEP
258 flaps (table 3). This finding corresponds to impaired nitric oxide-related cutaneous
259 vasodilation in response to acetylcholine iontophoresis reported in patients treated with
260 various chemotherapy regimens.²⁵,²⁶ Because of the protective effect of endogenous
261 estrogen on endothelial function²⁷, menopausal status, hormone replacement therapy,
262 and endocrine anticancer therapy are potentially relevant parameters. The study sample
263 showed little to no variation regarding menopausal status and hormone replacement
264 therapy. However, 11 participants (52%) received endocrine therapy, which, depending
on the mechanism of action, may either improve or reduce endothelial function.
Tamoxifen and related compounds competitively antagonize estrogen receptors and can
increase serum estrogen levels in postmenopausal women<sup>28</sup>, with potential protective
effects on endothelial function.<sup>29,30</sup> By contrast, aromatase inhibitors inhibit estrogen
production, reducing levels of endogenous estrogen and impairing endothelial
function.<sup>31,32</sup> In our study, chemotherapy likely confounded estimates of association
between tamoxifen and the magnitude of the vasodilator response. Lastly, advanced age
has been associated with significantly reduced peak and plateau skin blood flow in
response to local heating.<sup>33,34</sup> Absence of an inverse association between age and
microvascular function in this study might be attributable to the participants' narrow
range of age.

Several study limitations should be considered. Contralateral breasts are flawed controls
of abdomen-derived DIEP flaps. Although similar innervation densities have been
reported for female breast and abdominal skin³⁵, there could be important functional and
structural differences. For instance, a prior study reported significantly more pronounced
nitric oxide-dependent cutaneous vasodilation in response to local heating in the
abdomen than in the chest area.³⁶ Six of our study participants underwent surgical
procedures on their contralateral breast, which may have affected cutaneous nerve
integrity and microvascular function. For these reasons, we were unable to eliminate
potential patient-level confounding by expressing values as ratio to the contralateral
breast. Another study limitation is that blood flow recordings were not normalized to
maximal or baseline skin blood flow. Maximal values were not obtained, and baseline skin
blood flow was variable meaning that correction would have distorted rather than
rectified raw flux data. As site-to-site variability is perhaps the most important drawback
of the laser-Doppler technique³⁷, skin blood flow results should be interpreted with
caution and warrant follow-up study with adequate normalization.

The evidence derived from this study is too premature for informing clinical
recommendations regarding the effects of flap reinnervation on vasodilatory function.
293 The fact that we found no evidence for a better vasodilatory response to local heating in
294 reinnervated flaps does not mean that no such relationship exists. Further research is
295 needed to verify and build on our research findings before they can have wider
296 implications for clinical practice.

297 **CONCLUSION (87 words)**

298 This study suggests that long-term consequences of nerve disruption inherent to free flap
299 transfer extends beyond loss of sensory detection ability: we found significant
300 impairment of both the sensory nerve-mediated and nitric oxide-dependent local heating-
301 induced cutaneous vasodilatory systems that protect the skin from trauma. We found no
302 clear evidence that flap reinnervation enhances the skin's ability to mount an adequate
303 vasodilatory response to local heating. Nonetheless, significantly lower sensory
304 thresholds in reinnervated DIEP flaps suggest that reinnervation may provide a
305 meaningful degree of sensory nerve regeneration.
## ACKNOWLEDGEMENTS

We are grateful to our study participants for their considerable time and effort.
REFERENCES

1. Eltahir Y, Krabbe-Timmerman IS, Sadok N, Werker PMN, de Bock GH. Outcome of Quality of Life for Women Undergoing Autologous versus Alloplastic Breast Reconstruction following Mastectomy: A Systematic Review and Meta-Analysis. *Plast Reconstr Surg*. May 2020;145(5):1109-1123. doi:10.1097/PRS.00000000000006720

2. Bennett KG, Qi J, Kim HM, Hamill JB, Pusic AL, Wilkins EG. Comparison of 2-Year Complication Rates Among Common Techniques for Postmastectomy Breast Reconstruction. *JAMA Surg*. Oct 1 2018;153(10):901-908. doi:10.1001/jamasurg.2018.1687

3. Shammas RL, Fish LJ, Sergesketter AR, et al. Dissatisfaction After Post-Mastectomy Breast Reconstruction: A Mixed-Methods Study. *Ann Surg Oncol*. Feb 2022;29(2):1109-1119. doi:10.1245/s10434-021-10720-9

4. Delfino S, Brunetti B, Toto V, Persichetti P. Burn after breast reconstruction. *Burns*. Sep 2008;34(6):873-7. doi:10.1016/j.burns.2007.11.004

5. Enajat M, Rozen WM, Audolfsson T, Acosta R. Thermal injuries in the insensate deep inferior epigastric artery perforator flap: case series and literature review on mechanisms of injury. *Microsurgery*. 2009;29(3):214-7. doi:10.1002/micr.20601

6. Mohanna PN, Raveendran SS, Ross DA, Roblin P. Thermal injuries to autologous breast reconstructions and their donor sites--literature review and report of six cases. *J Plast Reconstr Aesthet Surg*. Mar 2010;63(3):e255-60. doi:10.1016/j.bjps.2009.06.040

7. Kellogg DL, Jr., Pergola PE, Piest KL, et al. Cutaneous active vasodilation in humans is mediated by cholinergic nerve cotransmission. *Circ Res*. Dec 1995;77(6):1222-8. doi:10.1161/01.res.77.6.1222

8. Klasson S, Svensson K, Wollmer P, Velander P, Svensson H. Blood flow dynamics and sensitivity in breasts after reconstruction with DIEP-flap. *J Plast Surg Hand Surg*. Dec 2014;68(6):407-11. doi:10.1097/000656X.2014.903194

9. Kellogg DL, Jr., Liu Y, Kosiba IF, O'Donnell D. Role of nitric oxide in the vascular effects of local warming of the skin in humans. *J Appl Physiol (1985)*. Apr 1999;86(4):1185-90. doi:10.1152/jappl.1999.86.4.1185

10. Minson CT, Berry LT, Joyner MJ. Nitric oxide and neurally mediated regulation of skin blood flow during local heating. *J Appl Physiol (1985)*. Oct 2001;91(4):1619-26. doi:10.1152/jappl.2001.91.4.1619

11. Smith CJ, Johnson JM. Responses to hyperthermia. Optimizing heat dissipation by convection and evaporation: Neural control of skin blood flow and sweating in humans. *Auton Neurosci*. Apr 2016;196:25-36. doi:10.1016/j.autneu.2016.01.002

12. Shiah E, Laikhter E, Comer CD, et al. Neurotization in Innervated Breast Reconstruction: A Systematic Review of Techniques and Outcomes. *J Plast Reconstr Aesthet Surg*. Jun 17 2022;doi:10.1016/j.bjps.2022.06.006

13. Rickham PP. Human Experimentation. Code of Ethics of the World Medical Association. Declaration of Helsinki. *Br Med J*. Jul 18 1964;2(5402):177. doi:10.1136/bmj.2.5402.177

14. Beugels J, Bijkerk E, Lataster A, Heuts EM, van der Hulst R, Tuinder SMH. Nerve Coaptation Improves the Sensory Recovery of the Breast in DIEP Flap Breast Reconstruction. *Plast Reconstr Surg*. Aug 1 2021;148(2):273-284. doi:10.1097/PRS.00000000000008160

15. Doll C, Durand R, Grulkey W, Sayer S, Olivotto I. Functional assessment of cutaneous microvasculature after radiation. *Radiother Oncol*. Apr 1999;51(1):67-70. doi:10.1016/s0167-8140(99)00022-5

16. Rodriguez-Fontan F, Reeves B, Tuano K, Colakoglu S, L DA, Banegas R. Tobacco use and neurogenesis: A theoretical review of pathophysiological mechanism affecting the outcome of peripheral nerve regeneration. *J Orthop*. Nov-Dec 2020;22:59-63. doi:10.1016/j.jor.2020.03.026

17. Kennedy JM, Zochodne DW. Impaired peripheral nerve regeneration in diabetes mellitus. *J Peripher Nerv Syst*. Jun 2005;10(2):144-57. doi:10.1111/j.1085-9489.2005.0010205.x

18. Allen RJ, Treece P. Deep inferior epigastric perforator flap for breast reconstruction. *Ann Plast Surg*. Jan 1994;32(1):32-8. doi:10.1097/00000637-199401000-00007

19. Spiegel AJ, Salazar-Reyes H, Izaddoost S, Khan FN. A novel method for neurotization of deep inferior epigastric perforator and superficial inferior epigastric artery flaps. *Plast Reconstr Surg*. Jan 2009;123(1):29e-30e. doi:10.1097/PRS.0b013e3181905564

20. Bell-Krotoski J, Weinstein S, Weinstein C. Testing sensibility, including touch-pressure, two-point discrimination, point localization, and vibration. *J Hand Ther*. Apr-Jun 1993;6(2):114-23. doi:10.1016/s0894-1130(12)80292-4
21. Magerl W, Treede RD. Heat-evoked vasodilatation in human hairy skin: axon reflexes due to low-level activity of nociceptive afferents. *J Physiol*. Dec 15 1996;497 ( Pt 3):837-48.
doi:10.1113/jphysiol.1996.sp021814

22. Glatte P, Buchmann SJ, Hijazi MM, Illigens BM, Siepmann T. Architecture of the Cutaneous Autonomic Nervous System. *Front Neurol*. 2019;10:970. doi:10.3389/fneur.2019.00970

23. Caselli A, Rich J, Hanane T, Uccioli L, Veves A. Role of C-nociceptive fibers in the nerve axon reflex-related vasodilation in diabetes. *Neurology*. Jan 28 2003;60(2):297-300.
doi:10.1212/01.wnl.0000040250.31755.f9

24. Pergola PE, Kellogg DL, Jr., Johnson JM, Kosiba WA, Solomon DE. Role of sympathetic nerves in the vascular effects of local temperature in human forearm skin. *Am J Physiol*. Sep 1993;265(3 Pt 2):H785-92. doi:10.1152/ajpheart.1993.265.3.H785

25. Fredslund SO, Buus NH, Hojgaard Skjold C, Laugesen E, Jensen AB, Laursen BE. Changes in vascular function during breast cancer treatment. *Br J Clin Pharmacol*. Nov 2021;87(11):4230-4240. doi:10.1111/bcp.14837

26. Sutterfield SL, Caldwell JT, Post HK, Lovoy GM, Banister HR, Ade CJ. Lower cutaneous microvascular reactivity in adult cancer patients receiving chemotherapy. *J Appl Physiol* (1985). Oct 1 2018;125(4):1141-1149. doi:10.1152/japplphysiol.00394.2018

27. Ganz P. Vasomotor and vascular effects of hormone replacement therapy. *Am J Cardiol*. Jul 3 2002;90(1A):11F-16F. doi:10.1016/s0002-9149(01)02218-4

28. Lum SS, Woltering EA, Fletcher WS, Pommier RF. Changes in serum estrogen levels in women during tamoxifen therapy. *Am J Surg*. May 1997;173(5):399-402.
doi:10.1016/S0002-9610(97)00072-X

29. Sutterfield SL, Caldwell JT, Post HK, Lovoy GM, Banister HR, Ade CJ. Cardiovascular effects of droloxifene, a new selective estrogen receptor modulator, in healthy postmenopausal women. *Arterioscler Thromb Vasc Biol*. Jun 2000;20(6):1606-12. doi:10.1161/01.atv.20.6.1606

30. Sarrel PM, Nawaz H, Chan W, Fuchs M, Katz DL. Raloxifene and endothelial function in healthy postmenopausal women. *Am J Obstet Gynecol*. Feb 2003;188(2):304-9.
doi:10.1067/mob.2003.28

31. Blaes A, Beckwith H, Florea N, et al. Vascular function in breast cancer survivors on aromatase inhibitors: a pilot study. *Breast Cancer Res Treat*. Nov 2017;166(2):541-547.
doi:10.1007/s10549-017-4447-6

32. Maor R, Sara JDS, Wanous AA, et al. Attenuated peripheral endothelial function among women treated with aromatase inhibitors for breast cancer. *Coron Artery Dis*. Dec 2018;29(8):687-693. doi:10.1097/MCA.0000000000000666

33. Minson CT, Holowatz LA, Wong BJ, Kenney WL, Wilkins BW. Decreased nitric oxide- and axon reflex-mediated cutaneous vasodilation with age during local heating. *J Appl Physiol* (1985). Nov 2002;93(5):1644-9. doi:10.1152/japplphysiol.00229.2002

34. Fromy B, Sigaudo-Roussel D, Gaubert-Dahan ML, et al. Aging-associated sensory neuropathy alters pressure-induced vasodilation in humans. *J Invest Dermatol*. Mar 2010;130(3):849-55. doi:10.1038/jid.2009.279

35. Besne I, Descombes C, Breton L. Effect of age and anatomical site on density of sensory innervation in human epidermis. *Arch Dermatol*. Nov 2002;138(11):1445-50.
doi:10.1001/archderm.138.11.1445

36. McGarr GW, King KE, Saci S, et al. Regional variation in nitric oxide-dependent cutaneous vasodilatation during local heating in young adults. *Exp Physiol*. Aug 2021;106(8):1671-1678. doi:10.1113/EP089671

37. Chaseling GK, Crandall CG, Gagnon D. Skin blood flow measurements during heat stress: technical and analytical considerations. *Am J Physiol Regul Integr Comp Physiol*. Jan 1 2020;318(1):R57-R69. doi:10.1152/ajpregu.00177.2019
TABLES

<table><caption>**Table 1.** Participant characteristics, by outcome group</caption><thead><tr><th></th><th>Reinnervated<br>DIEP flaps</th><th>Noninnervated<br>DIEP flaps</th></tr></thead><tbody><tr><td>No. of participants</td><td>10</td><td>11</td></tr><tr><td>Mean age at time of experiment, yr</td><td>55 ± 9</td><td>60 ± 12</td></tr><tr><td>Mean body mass index, kg/m<sup>2</sup></td><td>24 ± 2</td><td>25 ± 4</td></tr><tr><td>Menopausal status, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">Premenopausal</td><td>0</td><td>1 (9)</td></tr><tr><td style="padding-left: 20px;">Postmenopausal</td><td>10 (100)</td><td>11 (91)</td></tr><tr><td>Ex-smoker, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>5 (50)</td><td>5 (45)</td></tr><tr><td style="padding-left: 20px;">Yes</td><td>5 (50)</td><td>6 (55)</td></tr><tr><td>Small fiber neuropathy</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No, no. (%)</td><td>7 (70)</td><td>5 (45)</td></tr><tr><td style="padding-left: 20px;">Yes, no. (%)<sup>a</sup></td><td>3 (30)</td><td>6 (55)</td></tr><tr><td>Time since symptom onset, median (25<sup>th</sup>-75<sup>th</sup> percentile), yr</td><td>6 (5 - 7)</td><td>27 (18 - 35)</td></tr><tr><td>Hypertension, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>8 (80)</td><td>8 (73)</td></tr><tr><td style="padding-left: 20px;">Yes</td><td>2 (20)</td><td>3 (27)</td></tr><tr><td>Hypercholesterolemia, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>10 (100)</td><td>11 (91)</td></tr><tr><td style="padding-left: 20px;">Yes</td><td>0</td><td>1 (9)</td></tr><tr><td>Indication for mastectomy, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">Invasive breast cancer</td><td>10 (100)</td><td>9 (82)</td></tr><tr><td style="padding-left: 20px;">Ductal carcinoma in situ</td><td>0</td><td>2 (18)</td></tr><tr><td>Chemotherapy, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>2 (20)</td><td>4 (36)</td></tr><tr><td style="padding-left: 20px;">Yes<sup>b</sup></td><td>8 (80)</td><td>7 (64)</td></tr><tr><td>Trastuzumab, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>8 (80)</td><td>10 (91)</td></tr><tr><td style="padding-left: 20px;">Yes</td><td>2 (20)</td><td>1 (9)</td></tr><tr><td>Endocrine therapy, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">None</td><td>3 (30)</td><td>7 (64)</td></tr><tr><td style="padding-left: 20px;">Tamoxifen</td><td>6 (60)</td><td>3 (27)</td></tr><tr><td style="padding-left: 20px;">Anastrozole</td><td>1 (10)</td><td>1 (9)</td></tr><tr><td>Pre-reconstruction radiotherapy, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>6 (60)</td><td>8 (73)</td></tr><tr><td style="padding-left: 20px;">Yes</td><td>4 (40)</td><td>3 (27)</td></tr><tr><td>Flap type, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">Unipedicled DIEP flap</td><td>5 (50)</td><td>8 (73)</td></tr><tr><td style="padding-left: 20px;">Bipedicled or stacked DIEP flap</td><td>5 (50)</td><td>3 (27)</td></tr><tr><td>Recipient nerve used for coaptation</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">Anterior cutaneous branch of the second intercostal nerve</td><td>4 (40)</td><td>-</td></tr><tr><td style="padding-left: 20px;">Anterior cutaneous branch of the third intercostal nerve</td><td>6 (60)</td><td>-</td></tr><tr><td>Mean flap weight, g<sup>c</sup></td><td>610 ± 296</td><td>613 ± 169</td></tr><tr><td>Mean ischemia time, min</td><td>43 ± 8</td><td>47 ± 13</td></tr><tr><td>Mean follow-up time, yr</td><td>5 ± 1</td><td>5 ± 1</td></tr><tr><td>Contralateral breast symmetrization, no. (%)</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">No</td><td>6 (60)</td><td>9 (82)</td></tr><tr><td style="padding-left: 20px;">Yes<sup>d</sup></td><td>4 (40)</td><td>2 (18)</td></tr><tr><td>Mean core temperature before experiment, °C</td><td>36.6 ± 0.2</td><td>36.6 ± 0.2</td></tr><tr><td>Mean systolic blood pressure, mmHg</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">Before experiment</td><td>138 ± 25</td><td>131 ± 17</td></tr><tr><td style="padding-left: 20px;">After experiment</td><td>143 ± 26</td><td>137 ± 20</td></tr><tr><td>Mean diastolic blood pressure, mmHg</td><td></td><td></td></tr><tr><td style="padding-left: 20px;">Before experiment</td><td>92 ± 17</td><td>89 ± 12</td></tr><tr><td style="padding-left: 20px;">After experiment</td><td>94 ± 21</td><td>93 ± 12</td></tr></tbody></table>
Note: Plus-minus values are means ± SD.

Abbreviation: DIEP, deep inferior epigastric perforator.

<sup>a</sup> Related to chemotherapy (n = 5), sarcoidosis, Crohn's disease, Lyme disease, or vitamin B6 intoxication.

<sup>b</sup> I.e., doxorubicin, cyclophosphamide, paclitaxel, and docetaxel, in various combinations.

<sup>c</sup> For breast reconstructions involving two stacked flaps, only the heaviest flap was considered.

<sup>d</sup> I.e., breast reduction (n = 5) and breast augmentation (n = 1).
<table><caption>**Table 2.** Skin blood flow in response to local heating</caption><thead><tr><th></th><th>Contralateral<br>breasts</th><th>Reinnervated<br>DIEP flaps</th><th>Noninnervated<br>DIEP flaps</th><th>p</th></tr></thead><tbody><tr><td>No. of breasts/flaps</td><td>21</td><td>10</td><td>11</td><td></td></tr><tr><td>Mean skin temperature, °C</td><td>31.7 ± 1.4</td><td>32.0 ± 0.8</td><td>32.0 ± 1.0</td><td>0.66</td></tr><tr><td>Median baseline skin blood flow, (25<sup>th</sup>-75<sup>th</sup> percentile), a.u.</td><td>23 (15 - 38)</td><td>20 (13 - 35)</td><td>22 (14 - 26)</td><td>0.41</td></tr><tr><td>Mean onset of peak skin blood flow, min</td><td>2.8 ± 0.7</td><td>2.8 ± 0.5</td><td>3.0 ± 0.5</td><td>0.88</td></tr><tr><td>Median peak skin blood flow, (25<sup>th</sup>-75<sup>th</sup> percentile), a.u.</td><td>94 (74 - 141)</td><td>55 (39 - 68)</td><td>66 (36 - 77)</td><td>0.002</td></tr><tr><td>Median plateau skin blood flow, (25<sup>th</sup>-75<sup>th</sup> percentile), a.u.</td><td>145 (112 - 234)</td><td>71 (63 - 101)</td><td>86 (57 - 142)</td><td>0.004</td></tr><tr><td>Median area under the curve, (25<sup>th</sup>-75<sup>th</sup> percentile), (x10<sup>3</sup> a.u. x min)</td><td>126 (109 - 213)</td><td>82 (51 - 120)</td><td>81 (54 - 103)</td><td>0.003</td></tr></tbody></table>

Note: Plus-minus values are means ± SD. Baseline skin blood flow was calculated as average skin blood flow over a one-minute normothermic period.

Abbreviations: DIEP, deep inferior epigastric perforator; a.u., arbitrary units.
<table><caption>**Table 3.** Univariable linear regression analysis of factors associated with skin blood flow in response to local heating</caption><thead><tr><th rowspan="3"></th><th colspan="6">Contralateral breasts</th><th colspan="6">DIEP flaps</th></tr><tr><th colspan="3">Peak</th><th colspan="3">Plateau</th><th colspan="3">Peak</th><th colspan="3">Plateau</th></tr><tr><th>β</th><th>95% CI</th><th>p</th><th>β</th><th>95% CI</th><th>p</th><th>β</th><th>95% CI</th><th>p</th><th>β</th><th>95% CI</th><th>p</th></tr></thead><tbody><tr><td>Univariable analysis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Flap reinnervation</td><td></td><td></td><td></td><td></td><td></td><td></td><td>4</td><td>-26, 35</td><td>0.77</td><td>-9</td><td>-56, 39</td><td>0.71</td></tr><tr><td>Follow-up time, yr</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>-12, 13</td><td>0.94</td><td>5</td><td>15, 24</td><td>0.62</td></tr><tr><td>Chemotherapy</td><td>-40</td><td>-38, 3</td><td>0.07</td><td>-32</td><td>-114, 50</td><td>0.42</td><td>-14</td><td>-47, 20</td><td>0.40</td><td>-45</td><td>-93, 3</td><td>0.06</td></tr><tr><td>Tamoxifen</td><td>-37</td><td>-77, 2</td><td>0.06</td><td>-13</td><td>-89, 62</td><td>0.71</td><td>-5</td><td>-36, 26</td><td>0.74</td><td>-23</td><td>-70, 23</td><td>0.30</td></tr><tr><td>Hypertension</td><td>21</td><td>-28, 71</td><td>0.37</td><td>-13</td><td>-101, 75</td><td>0.76</td><td>-3</td><td>-40, 33</td><td>0.85</td><td>8</td><td>-47, 64</td><td>0.76</td></tr><tr><td>Ex-smoker</td><td>-13</td><td>-56, 29</td><td>0.52</td><td>-26</td><td>-100, 48</td><td>0.48</td><td>-2</td><td>-33, 29</td><td>0.90</td><td>6</td><td>-41, 54</td><td>0.78</td></tr><tr><td>Small fiber neuropathy</td><td>5</td><td>-38, 48</td><td>0.81</td><td>32</td><td>-42, 106</td><td>0.38</td><td>7</td><td>-24, 38</td><td>0.63</td><td>-1</td><td>-49, 46</td><td>0.95</td></tr><tr><td>Age at time of experiment, yr</td><td>2</td><td>-0, 4</td><td>0.07</td><td>-0</td><td>-4, 3</td><td>0.92</td><td>0</td><td>-1, 2</td><td>0.54</td><td>1</td><td>-1, 3</td><td>0.44</td></tr><tr><td>Contralateral breast symmetrization</td><td>-33</td><td>-78, 11</td><td>0.14</td><td>-26</td><td>-108, 57</td><td>0.52</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Abbreviations: DIEP, deep inferior epigastric perforator; CI, confidence interval.
<table><caption>**Table 4.** Warm detection and heat pain thresholds, by outcome group</caption><thead><tr><th></th><th>Reinnervated<br>DIEP flaps</th><th>Noninnervated<br>DIEP flaps</th></tr></thead><tbody><tr><td>No. of participants</td><td>10</td><td>11</td></tr><tr><td>WDT, no. (%)</td><td></td><td></td></tr><tr><td>≤ 50°C</td><td>6 (60)</td><td>3 (27)</td></tr><tr><td>> 50°C</td><td>4 (40)</td><td>8 (73)</td></tr><tr><td>HPT, no. (%)*</td><td></td><td></td></tr><tr><td>≤ 50°C</td><td>9 (90)</td><td>4 (36)</td></tr><tr><td>> 50°C</td><td>1 (10)</td><td>7 (64)</td></tr></tbody></table>

Abbreviations: DIEP, deep inferior epigastric perforator; WDT, warm detection threshold; HPT, heat pain threshold.

* *p* < 0.05, by Fisher's exact test.
FIGURE LEGENDS

<figure><img src="image_1.png" /><figcaption>**Figure 1.** Experimental setup for local heating-induced cutaneous vasodilatation</figcaption></figure>

Skin blood flow was monitored using laser-Doppler flowmetry (PeriFlux System 6000; Perimed, Stockholm, Sweden). Medially on both the DIEP flap and contralateral breast, we simultaneously measured skin blood flow in response to local heating at 42°C using a 457-PI probe (Perimed, Stockholm, Sweden), and control skin blood flow using a 440 probe (Perimed, Stockholm, Sweden).

<figure><img src="image_2.png" /><figcaption>**Figure 2.** (A-C) Skin blood flow in response to local heating, (D) area under the curve</figcaption></figure>

(A-C) Thick colored lines represent median skin blood flow in response to local heating for 21 contralateral breasts (A), 10 reinnervated deep inferior epigastric perforator (DIEP) flaps (B), and 11 noninnervated DIEP flaps (C). Shaded areas represent interquartile spans. Thin colored lines represent mean control skin blood flow. (D) Horizontal lines inside the boxes represent median area under the curve (AUC). Shaded areas represent interquartile spans. a.u., arbitrary units. \* *p* < 0.05.

<figure><img src="image_3.png" /><figcaption>**Figure 3.** (A) Mechanical detection, (B) warm detection, (C) and heat pain thresholds</figcaption></figure>
Horizontal lines inside the boxes represent median mechanical detection thresholds (MDT; A), warm detection thresholds (WDT; B), and heat pain thresholds (HPT; C) for 21 contralateral breasts, 10 reinnervated deep inferior epigastric perforator (DIEP) flaps, and 11 noninnervated DIEP flaps. Shaded areas represent interquartile spans. Red lines represent the 50°C safety limit. * *p* < 0.05.

<figure><img src="image_4.png" /><figcaption>**Figure 4.** Structural integrity of dermal and epidermal small sensory nerve fibers (A) in abdominal control skin, but not (B) in a reinnervated DIEP skin island</figcaption></figure>

Dermal/epidermal small sensory nerves (PGP9.5; red) and cell nuclei (DAPI; blue) under 40× magnification.
The white dashed lines indicate the dermal-epidermal junction.