Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review.

PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-ß cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells.

Case Report of Gas Gangrene after Reconstructive Surgery with Anterolateral Thigh Flap for Resection of Oral Cancer.

Necrotizing fasciitis (NF) type I is an acute subcutaneous tissue infection that can promptly disseminate generating crepitus. If not accurately diagnosed and expeditiously treated, it becomes a life-threatening infection. In this report, we present a 65-year-old man who developed a case of NF after a hemiglossectomy resecting a tumor in the dorsal surface of the tongue. A biopsy was performed, and he was pathologically diagnosed with squamous cell carcinoma (T2N1M0). The patient underwent preoperative oral cleaning. Right hemiglossectomy was performed by cervical dissection, pull-through style, with tooth removal on the right mandible and a left anterolateral femoral flap reconstruction. Routine intraoperative lavage was performed with 2000 mL of saline solution. Cefazolin 1gr was administered two times per day postoperatively. Four days after primary surgery, the flap circulation was inadequate; therefore, a computed tomography scan was taken, which indicated gas in the ventral neck area. Tooth extraction was the suspected etiology. Debridement was performed; the abscess was drained and cultured, indicating the presence of Staphylococcus haemolyticus and Escherichia coli. It seems that the abscess was not formed by NF, but rather by leachate reservoir associated with the head and neck tumor. After debridement, re-reconstruction was performed with a deltopectoral flap and pectoralis major myocutaneous flap. When NF is present after a neck dissection, there is a risk of disruption due to the direct invasion and inflammation into the carotid artery. Therefore, it is important to provide adequate oral cleaning care before the surgery and early suspicion of the diagnosis.

Clinical applications of the chimeric anterolateral thigh (ALT) flap in head and neck reconstruction.

BACKGROUND: Surgical site infection (SSI) is a common complication following head and neck surgery. Dead space at the excision site can increase the risk of infection, abscess formation, and mortality. Herein, we evaluated the performance of the chimeric anterolateral thigh (ALT) flap in addressing these concerns. METHODS: Patients who underwent oncologic head and neck reconstruction between October 2016 and November 2021 were divided in two groups: a normal ALT flap and a chimeric dead space filling (DSF) ALT-vastus lateralis flap group. We evaluated the postoperative outcomes. RESULTS: Twenty-five patients treated with normal ALT flaps (34.7%) and 47 with DSF ALT flap group (65.2%) were included. Only one ALT per case was necessary. Most of the cases involved tongue (31.9%) and lower gingival reconstruction (27.8%). The time to harvest the DSF ALT flap was 134.3 min when compared to the normal ALT flap (116.2 min, p < .001). Vascular occlusion, flap loss, partial necrosis, and fat necrosis were not observed among the different groups. CONCLUSION: The DSF process can be used as a preventive measure for SSI or vessel exposure due to radiation-induced skin damage. This flap allows same-site reconstruction if the primary tumor recurs by using the pedicle of the chimeric flap for reattachment of another free flap.

SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells.

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not-e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased ß3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

Characteristics and Limitations of Video-capillaroscopy in Reconstructive Microsurgery for Different Histologic Components of Flaps.

Indocyanine green, ultrasonography, and handheld Doppler can be used to evaluate blood flow at the donor and recipient site during microvascular reconstruction. However, these methods do not provide direct visualization and assessment of real-time blood flow. Video-capillaroscopy has been shown to be useful in clinical practice to assess microcirculation in rheumatologic disorders. In this report we used video-capillaroscopy to assess different tissue components involved in microvascular surgery. Seven patients who underwent head and neck oncologic microvascular reconstruction between November 2021 and February 2022 were included in this study. Video-capillaroscopy (GOKO-BscanZD, GOKO Imaging Devices Co., Ltd., Japan) was used to evaluate the donor-site and recipient-site tissue components. Optimal red blood cell movement was graded with a score of four, while no flow was graded with a score of 0. Seven myocutaneous flaps and seven recipient sites were evaluated. For the donor-site, our analysis demonstrated a significantly higher video-capillaroscopy quality for skin (3.43), adipose tissue (3.7) and perforators (3.7) when compared with muscle (0.429), muscle fascia (0.857), and de-epithelialized skin (1) (P < 0.001). For the recipient-site, a significantly higher video-capillaroscopy quality for skin (2.7), adipose tissue (3.5), and the periosteum (2.1) was noted when compared with muscle (0) (P < 0.001). Video-capillaroscopy efficiency is limited in the muscular component and injured (de-epithelialized) skin surface areas of flaps. Herein, we provide evidence that assessment of flap perfusion with video-capillaroscopy can be reliably achieved in the skin, periosteum, perforators, and adipose tissue. Video-capillaroscopy is expected to be applied for intraoperative real-time blood flow evaluation.

Intraoperative Identification of Perforator Spasm and Decreased Pulsation Using Video-capillaroscopy during Free Flap Harvest.

Temporary or prolonged vascular spasm can be appreciated when perivascular dissection is performed for microvascular reconstruction. Due to a lack of reliable assessment modalities, the resolution of spasm at the periphery of perforators cannot be determined by naked eyes or surgical loupes. To address this conundrum, we aimed to observe the state of perforator vessel spasm after flap elevation using video-capillaroscopy. Between November 2021 and February 2022, seven free flaps were evaluated with video-capillaroscopy to determine the incidence of vasospasm in less than 1 mm-diameter perforators. The type of perforator spasm after flap elevation was divided into six types according to the video-capillaroscopy findings: type A, no spasm/decreased pulsation (S/DP); type B, S/DP with recovery within 5 minutes; type C, S/DP requiring papaverine hydrochloride spraying and hot water treatment (PHS+HWT) resulting in recovery within 5 minutes; type D, S/DP requiring PHS+HWT resulting in recovery within 10 minutes; type E, S/DP requiring PHS+HWT resulting in recovery within 15 minutes; and type F, S/DP with no recovery of pulsation even after PHS+HWT. Twenty-five perforators were evaluated, 3.57 perforators (range, 3-4) per flap. Using our classification for perforator vessel spasms on video-capillaroscopy, observations of five perforating branches were classified as type A, seven as type B, six as type C, five as type D, and two as type E. No type F spasm was observed. With video-capillaroscopy it is possible to confirm if blood flow deterioration occurs even in areas that are difficult to determine macroscopically. Video-capillaroscopy, a noninvasive imaging modality, is a useful alternative for the intraoperative evaluation of perforator flow and spasm.

Dental Silicone-based Surgical Guides to Harvest the Chimeric Scapular Flap: Preventing Iatrogenic Vascular Injury.

The angular branch of the thoracodorsal artery and the periosteal branches of the circumflex scapular artery can be easily injured while harvesting a chimeric scapular flap. Thus, we reported the use of 3D printed scapular models using CT angiography to prepare inexpensive surgical guides from autoclavable dental silicone impressions for scapular flap harvest. Mandibular and scapular models were prepared using a 3D printer for 11 patients undergoing chimeric scapular flap transfer following mandibular resection. During preoperative simulation surgery, we molded dental silicone accordingly with scapular models to produce surgical cutting guides. Six men (54.5%) and five women (45.5%) were included. The average age of patients was 65.4 years. Fourteen bone units were reconstructed as three patients needed two bone segments (27.3%) whereas eight patients required reconstruction of one bone segment (72.7%). The mean flap harvest time and total surgical time were 52.1 min and 633.8 min, respectively. The mean duration for osteotomies and bone plate fixation was 26.2 min. The difference between the length of the preoperative surgical model (64.92 mm) and the postoperative 3D-CT measurements (64.48 mm) was not statistically significant (0.95 mm, P = 0.397). No injuries were caused to the angular and periosteal vessels. Four patients exhibited donor-site seroma (36.4%). The cost of the dental silicone for surgical guide was only $5 per patient. Dental silicone-based surgical guides help minimize the risk of vascular injury while harvesting chimeric scapular flaps. The osteotomies were performed with precision and in a time-efficient manner.

Real-Time Assessment of Free Flap Capillary Circulation Using Videocapillaroscopy.

BACKGROUND: Free tissue transfer is a common method of reconstruction for various surgical defects. Many clinical assessment methods, including flap color monitoring and capillary refill time measurements, are commonly used to assess the microcirculation of the flap, yet there is no objective tool available that can clear real-time visualization of the flap microcirculation. The purpose of this study was to use a novel videocapillaroscope to evaluate the circulation changes on free flap skin surfaces while purposely clamping pedicle vessels. METHODS: Ten patients who underwent free flap transfer for head and neck cancer from November of 2019 to June of 2020 were included in the study. Videocapillaroscopic observation was performed after flap elevation, and changes in the flap skin capillary circulation with artery-controlled and vein-controlled clamping were recorded. RESULTS: Average total surgery time was 517.91 minutes (SD, 73.3 minutes), average flap elevation time was 102.9 minutes (SD, 18.3 minutes). When the pedicle artery or vein was purposely clamped, the movement of red blood cells in blood vessels stopped; when clamps were removed after 60 seconds, the restoration of red blood cell movements was rapidly observed. When the pedicle artery was clamped, the number of visualizable blood vessels decreased and flap color became relatively white. When the pedicle vein was clamped, the number of visualizable blood vessels increased, and flap color tone had a tendency toward red. CONCLUSION: Novel videocapillaroscopy can be utilized for objective real-time flap monitoring by directly visualizing flap skin capillary microcirculation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Dynamic Changes on Video-capillaroscopy and Potential Microsurgical Application: Temperature and Skin Surface Readings.

Video-capillaroscopy is being explored as a potential tool for microsurgical flap monitoring. A detailed examination of the effect of temperature on capillary changes using video-capillaroscopy is yet to be investigated. We analyzed the video-capillaroscopy findings on different skin areas often used for flap harvest at normal body temperatures and at lower temperatures. Skin capillaries at the lateral thigh, anterior forearm, mid-axillary line, abdomen, and fingertips were observed using video-capillaroscopy in 20 healthy Japanese individuals. Further, ImageJ software was used to measure the blood vessel area and blood flow velocity, and comparisons were drawn between normal body temperature and lower body temperature states. All measures of blood vessel area and average blood flow velocity for the different anatomical regions were significantly different before and after cooling (P < 0.001). The mean reduction rate of the vessel area was significantly different among anatomic regions (P < 0.001). Post-hoc analysis revealed a significant difference in the vessel area reduction rate between anatomic areas (P < 0.05); except when comparing the thigh versus finger, the forearm versus abdomen, and the mid-axillary line versus abdomen. The mean blood flow velocity was significantly different among anatomic regions (P < 0.001). Post-hoc analysis revealed a significant difference between the thigh and forearm (P = 0.009), the forearm and fingertip (P = 0.001), and the abdomen and fingertip (P = 0.004). Decreasing the skin temperature resulted in a significant vasoconstriction and reduction in capillary flow velocity. It is imperative to keep the monitored area warm during video-capillaroscopy assessment to avoid false diagnosis of vascular occlusion.