Altered actin dynamics is possibly implicated in the inhibition of mechanical stimulation-induced dermal fibroblast differentiation into myofibroblasts.

The formation of hypertrophic scars and keloids is strongly associated with mechanical stimulation, and myofibroblasts are known to play a major role in abnormal scar formation. Wounds in patients with neurofibromatosis type 1 (NF1) become inconspicuous and lack the tendency to form abnormal scars. We hypothesized that there would be a unique response to mechanical stimulation and subsequent scar formation in NF1. To test this hypothesis, we investigated the molecular mechanisms of differentiation into myofibroblasts in NF1-derived fibroblasts and neurofibromin-depleted fibroblasts and examined actin dynamics, which is involved in fibroblast differentiation, with a focus on the pathway linking LIMK2/cofilin to actin dynamics. In normal fibroblasts, expression of α-smooth muscle actin (α-SMA), a marker of myofibroblasts, significantly increased after mechanical stimulation, whereas in NF1-derived and neurofibromin-depleted fibroblasts, α-SMA expression did not change. Phosphorylation of cofilin and subsequent actin polymerization did not increase in NF1-derived and neurofibromin-depleted fibroblasts after mechanical stimulation. Finally, in normal fibroblasts treated with Jasplakinolide, an actin stabilizer, α-SMA expression did not change after mechanical stimulation. Therefore, when neurofibromin was dysfunctional or depleted, subsequent actin polymerization did not occur in response to mechanical stimulation, which may have led to the unchanged expression of α-SMA. We believe this molecular pathway can be a potential therapeutic target for the treatment of abnormal scars.

"Hydrogen-generating Si-based agent improves fat graft survival in rats".

BACKGROUND: Regulating excessive inflammation and oxidative stress in fat grafting may improve retention rates. Hydrogen effectively combats oxidative stress and inflammation and reportedly inhibits ischemia-reperfusion injury in various organs. Unfortunately, with conventional methods of hydrogen administration, incorporating hydrogen continuously into the body over a long period of time is difficult. We hypothesized that a Silicon (Si)-based agent we recently developed would aid in fat grafting as it can generate large amounts of hydrogen continuously in the body. METHODS: Fat grafting was performed on the backs of rats fed either a normal or 1.0 wt% Si-based agent-containing diet. To investigate synergistic effects with adipose-derived stromal cells (ASCs), which improve retention rates of fat grafting, fat grafting with ASCs (1.0×10 5/400 mg fat) was also performed in each rat. Postoperative retention rates of grafted fat over time, inflammatory indices, apoptosis and oxidative stress markers, histological findings, and expression levels of inflammation-related cytokines and growth factors were compared between the four groups. RESULTS: Intake of Si-based agent and addition of ASCs significantly reduced inflammatory indices, oxidative stress, and apoptosis of grafted fat, and improved long-term retention rates, histological parameters, and grafted fat quality. Under our experimental conditions, intake of the Si-based agent and addition of ASCs yielded comparable improvements in fat graft retention. Combining the two enhanced these effects even further. CONCLUSION: Oral administration of the hydrogen-generating Si-based agent may improve grafted fat retention by regulating the inflammatory response and oxidative stress in grafted fat. CLINICAL RELEVANCE STATEMENT: This study demonstrates improved grafted fat retention rates using a Si-based agent. This Si-based agent has the potential to expand the range of therapeutic indications of hydrogen-based therapy to conditions for which hydrogen has yet to be found effective, such as fat grafting.

Lymphaticovenular Anastomosis for Lymphorrhea after Expander-based Breast Reconstruction: A Case Report.

Refractory axillary lymphorrhea is a postoperative complication of breast cancer with no established standard treatment. Recently, lymphaticovenular anastomosis (LVA) was used to treat not only lymphedema but also lymphorrhea and lymphocele in the inguinal and pelvic regions. However, only a few reports have been published on the treatment of axillary lymphatic leakage with LVA. This report presents a case of successful treatment of refractory axillary lymphorrhea after breast cancer surgery with LVA. A 68-year-old woman underwent nipple-sparing mastectomy for right breast cancer, axillary lymph node dissection, and immediate subpectoral tissue expander placement. Postoperatively, the patient developed refractory lymphorrhea and subsequent seroma around the tissue expander, and underwent postmastectomy radiation therapy and frequent percutaneous aspiration of the seroma. However, lymphatic leakage persisted, and surgical treatment was planned. Preoperative lymphoscintigraphy showed lymphatic outflow from the right axilla to the space around the tissue expander. There was no dermal backflow in the upper extremities. To reduce lymphatic flow into the axilla, LVA was performed at two sites in the right upper arm. The lymphatic vessels used for anastomosis were 0.35 mm and 0.50 mm in diameter, and each was anastomosed to the vein in an end-to-end fashion. The axillary lymphatic leakage stopped shortly after the operation, and there were no postoperative complications. LVA may be a safe and simple option for the treatment of axillary lymphorrhea.

Hydrogen-generating Si-based agent protects against skin flap ischemia-reperfusion injury in rats.

Hydrogen is effective against ischemia-reperfusion (I/R) injury in skin flaps. However, the difficulty of continuously administering a sufficient amount of hydrogen using conventional methods has been an issue in the clinical application of hydrogen-based therapy. An Si-based agent administered orally was previously shown to continuously generate a large amount of hydrogen in the intestinal environment. In this study, we assessed the effect of the Si-based agent on the inhibition of I/R injury in skin flaps using a rat model. In the I/R groups, the vascular pedicle of the abdominal skin flap was occluded for three hours followed by reperfusion. In the I/R + Si group, the Si-based agent was administered perioperatively. After reperfusion, flap survival rate, blood flow, oxidative stress markers, inflammatory markers/findings, and degree of apoptosis were evaluated. Flap survival rate was significantly higher, and histological inflammation, apoptotic cells, oxidative stress markers, and levels of inflammatory cytokine mRNA and protein expression were significantly lower, in the I/R + Si group compared to the I/R group. The Si-based agent suppressed oxidative stress, apoptosis, and inflammatory reactions resulting from I/R injury, thereby contributing to improvements in skin flap survival.

Effects of Temporary and Permanent Muscle Denervation on Fat Graft Retention in the Latissimus Dorsi Muscle: An Experimental Study in Rats.

BACKGROUND: In breast reconstruction with a latissimus dorsi flap, immediate fat grafting is useful for increasing flap volume. However, factors such as latissimus dorsi muscle atrophy and fat graft retention affect the volume of the reconstructed breast, and reports are inconsistent regarding treatment of the thoracodorsal nerve. This study examined how thoracodorsal nerve treatment affects the rates of latissimus dorsi flap preservation and fat graft retention using a rat model. METHODS: Fat harvested from the inguinal region was grafted to the latissimus dorsi muscle elevated as a pedicled muscle flap on the experimental side and to the intact LD muscle on the contralateral side (control). Rats were divided into intact thoracodorsal nerve (Ni), temporary denervation (Ntd), and permanent denervation (Npd) groups (n = 8 each). Fat retention and muscle preservation rates were determined, and histological changes were analyzed postoperatively. RESULTS: Fat retention rates did not significantly differ between the Ni and Ntd groups. Only the Npd group showed a significant decrease in fat retention rate relative to the control side (p < 0.01). The quality of the grafted fat as reflected by histological parameters was significantly lower, and the viable adipocyte area and muscle fiber preservation rate significantly decreased, in the Npd group compared to the other groups. CONCLUSIONS: Permanent thoracodorsal nerve denervation resulted in severe muscle atrophy and a significantly decreased fat retention rate. Temporary denervation had no significant benefit, suggesting that preserving the thoracodorsal nerve may be desirable for achieving sufficient volume in latissimus dorsi flap breast reconstruction with immediate fat grafting. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Sensory Reinnervation With Subcutaneously Embedded Innervated Flaps: An Experimental Study in Rats.

BACKGROUND: In breast reconstruction, both aesthetic outcomes and sensory function are important for postoperative quality of life. Innervated flaps are useful in reconstruction after conventional mastectomy (CM), which leaves a large portion of the skin paddle exposed on the body surface. However, whether they are also useful in skin-sparing mastectomy (SSM) and nipple-sparing mastectomy (NSM) remains unclear. This study aimed to examine the usefulness of innervated flaps in restoring sensation after SSM and NSM using a rat model. METHODS: Dorsal cutaneous nerves of rats were entirely eliminated except for the medial branch of the dorsal cutaneous nerve of thoracic segment 13, resulting in an innervated field surrounded by a denervated field. The innervated field was elevated as an innervated island flap and then subcutaneously embedded, with the skin paddle deepithelialized entirely (NSM group, n = 5) or except at the center (SSM group, n = 6). In the control model (CM group, n = 5), the flap was sutured back into its original position. Postoperative changes in the mechanonociceptive field were evaluated using the cutaneous trunci muscle reflex test. Immunohistochemical evaluation of regenerated nerves in the new mechanonociceptive field was performed at postoperative week 12. RESULTS: In the SSM and CM groups, the mechanonociceptive field expanded around the skin paddle. In the NSM group, a new mechanonociceptive field appeared at postoperative week 4 and expanded thereafter. Areas of the mechanonociceptive field at postoperative week 12 did not differ significantly between the SSM and NSM groups, but were significantly smaller compared with the CM group and comparable to original flap areas. Histologically, S100- and PGP9.5-positive nerve fibers were observed in the dermis of the new mechanonociceptive field and subcutaneous flap tissue. CONCLUSIONS: Subcutaneously embedded innervated island flaps induced nerve regeneration and sensory reinnervation of the denervated skin, suggesting that innervated flaps may also be useful in reconstruction post-SSM/NSM.

Targeted sensory reinnervation by direct neurotization of skin: An experimental study in rats.

BACKGROUND: No effective methods currently exist for breast neurotization in implant-based breast reconstruction. Here, we focused on direct neurotization (DN), in which axons regenerating from nerve stumps are directed to the mastectomy flap and aimed to assess whether DN can generate a new mechano-nociceptive field using a rat model of back skin sensory denervation. METHODS: Dorsal cutaneous nerves (DCNs) of rats were exposed and transected, leaving only the left medial branch of the DCN of thoracic segment 13 (mDCN-T13) intact. This procedure resulted in an isolated innervated field surrounded by a denervated field. The mDCN-T13 was transected, and the proximal nerve stump was sutured to the subdermis (DN subdermal group, n = 6) or dermis (DN dermal group, n = 5) of a different region of the denervated field. In the Crush group (n = 5), the intact mDCN-T13 was only crushed. We evaluated the generation of a new mechano-nociceptive field over time using the cutaneous trunci muscle (CTM) reflex test and histomorphometrically evaluated regenerating nerves in the reinnervated region. RESULTS: In the DN groups, the CTM reflex appeared in the DN area after postoperative week 4. The new mechano-nociceptive field gradually expanded afterwards, and by postoperative week 12, the area was substantially larger than the original region innervated by the mDCN-T13 in the DN dermal group, although not as large as that in the Crush group. In histomorphometric evaluations, many S100-positive myelinated fibers were observed in the dermis of the reinnervated area for all groups. CONCLUSION: In targeted sensory reinnervation, DN of the skin is revolutionary in that it allows a new innervated area to be generated at a desired location regardless of whether a distal nerve stump is available. DN may present an effective approach for breast neurotization in breast reconstruction after mastectomy, particularly for procedures that cannot use sensate flaps such as implant-based breast reconstruction.