Topical Insulin Eye Drops: Stability and Safety of Two Compounded Formulations for Treating Persistent Corneal Epithelial Defects.

Compounded insulin eye drops were prepared at 1 IU/mL from commercially available subcutaneous insulin by dilution in saline solution or artificial tears. Physicochemical characterization and in vitro tolerance testing in human and conjunctival cells were followed by a 28-day short-term stability study under various conditions. The formulations were isotonic (280-300 mOsm/L), had a pH close to neutral (7-8), medium surface-tension values (<56 MN/m-1), and low (≈1 mPa·s) and medium (≈5 mPa·s) viscosities (compounded normal saline solution and artificial tear-based preparation, respectively). These values remained stable for 28 days under refrigeration. Microbiological stability was also excellent. Insulin potency remained in the 90-110% range in the compounded formulations containing normal saline solution when stored at 2-8 °C for 28 days, while it decreased in those based on artificial tears. Although both formulations were well tolerated in vitro, the compounded insulin diluted in a normal saline solution exhibited better cell tolerance. Preliminary data in humans showed that insulin in saline solution was an effective and safe treatment for persistent corneal epithelial defects. Compounded insulin eye drops diluted in normal saline solution could, therefore, constitute an emergent therapy for the treatment of persistent corneal epithelial defects.

The role of topical insulin in ocular surface restoration: A review.

Corneal epithelial defects are one of the most common ocular disorders. Restoring corneal integrity is crucial to reduce pain and regain function, but in cases of neurotrophic or desensitized corneas, healing can be significantly delayed. Treating neurotrophic corneas is challenging for ophthalmologists, and surgical intervention is often indicated to manage refractory cases that are unresponsive to medical therapy. Over the last decade, as more expensive therapeutics reach the market, topical insulin has returned to the forefront as an affordable option to improve corneal wound healing. There is still a paucity of data on the use and the efficacy of topical insulin, with no consensus regarding its indications, preparation, or posology. Here we review the literature on topical insulin for corneal and ocular surface pathologies, with a focus on the current evidence, its mechanisms of action, and its safety profile. Additionally, we share our experience in the field and provide a potential framework for future research.

In Vivo Wound-Healing Efficacy of Insulin-Loaded Strontium-Cross-Linked Alginate-Based Hydrogels in Diabetic Rats.

The wound-healing effect of insulin is well studied and reported. However, prolonged topical application of insulin without compromising its biological activity is still a challenge. In this study, the effect of topically delivered insulin on promoting wound healing in diabetic animals was evaluated. Alginate diamine PEG-g-poly(PEGMA) (ADPM2S2) was the material used for the topical delivery of insulin. ADPM2S2 hydrogels release insulin and strontium ions, and they synergistically act to regulate different phases of wound healing. Insulin was released from the ADPM2S2 hydrogel for a period of 48 h, maintaining its structural stability and biological activity. In vitro studies were performed under high-glucose conditions to evaluate the wound-healing potential of insulin. Insulin-loaded ADPM2S2 hydrogels showed significant improvement in cell migration, proliferation, and collagen deposition, compared to control cells under high-glucose conditions. Immunostaining studies in L929 cells showed a reduction in phospho Akt expression under high-glucose conditions, and in the presence of insulin, the expression increased. The gene expression studies revealed that insulin plays an important role in regulating the inflammatory phase and macrophage polarization, which favors accelerated wound closure. In vivo experiments in diabetic rat excision wounds treated with insulin-loaded ADPM2S2 showed 95% wound closure within 14 days compared with 82% in control groups. Thus, both the in vitro and in vivo results signify the therapeutic potential of topically delivered insulin in wound management under high-glucose conditions.

Topical Insulin for Ocular Surface Disease.

Background: Insulin and insulin-like growth factor (IGF)-1 receptors are present in ocular tissues such as corneal epithelium, keratocytes, and conjunctival cells. Insulin plays a crucial role in the growth, differentiation, and proliferation of corneal epithelial cells, as well as in wound healing processes in various tissues. Purpose: This review explores the potential role of topical insulin in the treatment of ocular surface diseases. Specifically, it examines its impact on corneal nerve regeneration, sub-basal plexus corneal nerves, and its application in conditions like corneal epithelial defects, dry eye disease, and diabetic keratopathy. Methods: The review analyzes studies conducted over the past decade that have investigated the use of topical insulin in ocular surface diseases. It focuses on indications, drug preparation methods, side effects, efficacy outcomes, and variations in insulin concentrations and dosages used. Results: While off-label use of topical insulin has shown promising results in refractory corneal epithelial defects, its efficacy in dry eye disease is yet to be demonstrated. Variations in concentrations, dilutions, and dosing guidelines have been reported. However, limited data on ocular penetration, ocular toxicity, and systemic side effects pose challenges to its widespread utility. Conclusion: This review synthesizes findings from ocular investigations on topical insulin to assess its potential applicability in treating ocular surface and corneal diseases. By highlighting indications, preparation methods, side effects, and efficacy outcomes, it aims to provide insights into the current status and future prospects of using topical insulin in ophthalmic practice.

Insulin eye drops for refractory corneal epithelial defects. A case series / Colirio de insulina para defectos corneales epiteliales persistentes. Una serie de casos

Objetive: To report the clinical course of a case series of patients with persistent epithelial corneal defects (PECD) treated with insulin eye drops. Method: Retrospective review of five patients -four non-diabetic and one diabetic- in treatment with insulin eye drops 1 U/mL four times a day (QID). Results: Patients developed refractory epithelial ulcers due to different etiology (three infections, one trauma and one chemical injury). After treatment with topical insulin all defects were healed in about 30-60 days. Conclusion: Insulin formulated as 1 U/mL eye drops and administered QID can be an effective and safe option for PECD. (AU)

Objetivo: Comunicar la evolución clínica de una serie de casos de pacientes con defectos corneales epiteliales persistentes (PECD) tratados con colirio de insulina. Método: Revisión retrospectiva de cinco pacientes –cuatro no diabéticos y uno diabético– en tratamiento con colirio de insulina 1 U/mL cuatro veces al día (QID). Resultados: Los pacientes desarrollaron úlceras epiteliales refractarias de diferente etiología (tres infecciones, un traumatismo y una lesión química). Tras el tratamiento con insulina tópica todos las lesiones se curaron en unos 30-60 días. Conclusión: La insulina formulada en forma de colirio de 1 U/mL y administrada QID puede ser una opción eficaz y segura para la PECD. (AU)

Insulina colirio en afecciones de la superficie ocular

Cuando se produce una erosión corneal y fracasa la epitelización corneal surgen los defectos epiteliales corneales persistentes, cuyo tratamiento es un desafío para el oftalmólogo. Es muy frecuente el fracaso del tratamiento convencional por lo que se mantiene el interés en la búsqueda de otros factores de crecimiento para la cicatrización epitelial tales como los colirios de insulina. La insulina es un péptido estrechamente relacionado con el factor de crecimiento similar a la insulina 1. Su mecanismo de acción no es bien comprendido, sin embargo se acepta que es capaz de inducir migración y proliferación de las células epiteliales corneales, por lo que promueve y acelera la reepitelización de defectos epiteliales persistentes refractarios a tratamiento. La ausencia de una presentación comercial de colirio de insulina, hace necesario conocer su estabilidad físicoquímica y microbiológica así como la eficacia, efectividad y seguridad del colirio de insulina a diferentes concentraciones. De ahí la motivación para realizar una revisión de la literatura existente sobre el empleo del colirio de insulina en el tratamiento del defecto epitelial corneal persistente. Se realizó la búsqueda en bases de datos electrónicas como PubMed Central, EBSCO, Clinical Trials.gov, MEDLINE OVID, EMBASE OVID con el objeto de identificar artículos relacionados con el tema.

When corneal erosion occurs and corneal epithelialization fails, persistent corneal epithelial defects arise, whose treatment is a challenge for the ophthalmologist. The failure of conventional treatment is very frequent; therefore, there is still interest in the search for other growth factors for epithelial healing, such as insulin eye drops. Insulin is a peptide closely related to insulin-like growth factor 1. Its mechanism of action is not well understood; however, it is accepted that it is capable of inducing migration and proliferation of corneal epithelial cells, thereby promoting and accelerating reepithelialization of persistent epithelial defects refractory to treatment. The absence of a commercial presentation for insulin eye drops makes it necessary to know its physicochemical and microbiological stability, as well as the efficacy, effectiveness and safety of insulin eye drops at different concentrations; hence the motivation to review the existing literature on the use of insulin eye drops in the treatment of persistent corneal epithelial defects. The search was carried out in electronic databases such as PubMed Central, EBSCO, Clinical Trials.gov, MEDLINE OVID, EMBASE OVID, with the aim of identifying relevant articles related to the topic.

Neurotrophic Keratopathy and Topical Insulin Therapy: A Case Report.

Neurotrophic keratopathy is a rare disorder caused by the loss of corneal sensation. It is characterized by persistent epithelial defects, corneal ulceration, and, ultimately, corneal perforation if not managed in a timely manner. The management includes aggressive lubrication, prophylactic topical antibiotics, therapeutic contact lenses, tarsorrhaphy, and amniotic membrane transplantation. Some novel therapeutic options are also available, one of which is topical insulin therapy. We report the clinical course of a patient with neurotrophic keratopathy that was successfully treated with topical insulin. A 64-year-old male presented to our outpatient department with a three-month history of painless blurring of vision following prior episodes of herpetic keratitis. Ocular examination showed a bilateral reduction in corneal sensations, bilateral corneal opacities, and a corneal ulcer in the left eye. He was diagnosed as a case of neurotrophic keratopathy secondary to prior herpetic keratitis. He was then treated with topical and oral acyclovir along with topical insulin drops. There was a remarkable improvement in his condition after a month with a reduction in the size of the ulcer and, after two months, the ulcer was completely re-epithelialized. This case report illustrates the use of topical insulin in the initial management of neurotrophic keratopathy as opposed to its conventional use in refractory neurotrophic corneal ulcers.

Usage of topical insulin for the treatment of diabetic keratopathy, including corneal epithelial defects.

BACKGROUND: Diabetic keratopathy (DK) occurs in 46%-64% of patients with diabetes and requires serious attention. In patients with diabetes, the healing of corneal epithelial defects or ulcers takes longer than in patients without diabetes. Insulin is an effective factor in wound healing. The ability of systemic insulin to rapidly heal burn wounds has been reported for nearly a century, but only a few studies have been performed on the effects of topical insulin (TI) on the eye. Treatment with TI is effective in treating DK. AIM: To review clinical and experimental animal studies providing evidence for the efficacy of TI to heal corneal wounds. METHODS: National and international databases, including PubMed and Scopus, were searched using relevant keywords, and additional manual searches were conducted to assess the effectiveness of TI application on corneal wound healing. Journal articles published from January 1, 2000 to December 1, 2022 were examined. The relevancy of the identified citations was checked against predetermined eligibility standards, and relevant articles were extracted and reviewed. RESULTS: A total of eight articles were found relevant to be discussed in this review, including four animal studies and four clinical studies. According to the studies conducted, TI is effective for corneal re-epithelialization in patients with diabetes based on corneal wound size and healing rate. CONCLUSION: Available animal and clinical studies have shown that TI promotes corneal wound healing by several mechanisms. The use of TI was not associated with adverse effects in any of the published cases. Further studies are needed to enhance our knowledge and understanding of TI in the healing of DK.

Efficacy of topical insulin for recurrent epithelial corneal erosions.

BACKGROUND: Topical insulin can promote and accelerate corneal regeneration, even in eyes with serious comorbidities, and offers several benefits over other treatment options. AIMS: The aim of this study is to evaluate the effect of topical insulin in treatment of recurrent epithelial corneal erosion. METHODS: Patients with recurrent epithelial erosions were included in a prospective non-randomized hospital-based study, divided into two groups, one of them received persistent epithelial defects (PEDs) conventional treatment and the other received the same treatment with insulin eye drops 4 times/day. All patients were examined carefully by slit lamp. Patients during the 1st, 2nd, 3rd, and 4th weeks as well as after 2 months. Demographics, etiology, therapy, comorbidities, and the healing time of PED were performed. RESULTS: Area shows significant improvement after 2 weeks (p = 0.006), 2 months (p = 0.046), and 3 months (p = 0.002) in group II (cornetears gel and topical insulin) as compared to group I (cornetears gel). The recurrence was statistically significant decreased with cornetears gel and topical insulin (group II) by 0.0%, as compared to cornetears gel (group I) by 3 patients (21.4%). CONCLUSION: Topical insulin can promote corneal reepithelization in recurrent epithelial erosion and decreases recurrence in these cases. Other advantages include excellent tolerance, availability, and cost-effectiveness.

Efectividad de la insulina tópica en el tratamiento de patologías de la superficie corneal / Effectiveness of topical insulin for the treatment of surface corneal pathologies

El propósito es identificar a través de una revisión sistemática de la literatura, la evidencia actual frente a la eficacia del tratamiento de la insulina tópica en patologías de la superficie ocular. Se implementó una búsqueda de literatura en bases de datos de indexación médica Medline (Pubmed), Embase y Web Of Science a través de palabras claves como «insulin» AND «córnea» OR «corneal» OR «dry eye» artículos publicados en inglés o español en los últimos once años (2011-2022). Se identificaron nueve artículos con 180 participantes provenientes de Estados Unidos, España, Irlanda, Canadá, Portugal y Malasia, con defectos epiteliales persistentes refractarios y secundarios a vitrectomía, cuya extensión de la lesión fue de 3,75 mm2 hasta 65,47 mm2. La preparación fue disuelta con lágrimas artificiales y la concentración de insulina fue desde 1 UI/ml hasta 100 UI/ml. En todos los casos la resolución del cuadro clínico fue completa con un tiempo de curación desde 2,5 días hasta 60,9 días siendo este último un caso secundario a una quemadura por cáusticos de difícil control. La insulina tópica ha sido efectiva para el tratamiento de defectos epiteliales persistentes; la de acción intermedia y en bajas concentraciones demostró menor tiempo de resolución, en úlceras neurotróficas y secundarias a vitrectomías (AU)

The purpose is to identify, through a systematic literature review, the current evidence regarding the effectiveness of topical insulin treatment in ocular surface pathologies. A literature search was implemented in Medline (Pubmed), Embase and Web Of Science medical indexing databases by using keywords such as “insulin” AND “cornea” OR “corneal” OR “dry eye” in published papers in English or Spanish within the last eleven years (2011-2022). Nine papers were identified with 180 participants from the United States, Spain, Ireland, Canada, Portugal and Malaysia, with persistent refractory epithelial defects and secondary to vitrectomy, whose extension of the lesion was from 3.75 mm2 to 65.47 mm2. The preparation was dissolved with artificial tears and the insulin concentration ranged from 1 IU/ml to 100 IU/ml. In all cases, the resolution of the clinical picture was complete with a healing time from 2.5 days to 60.9 days, the latter being a secondary case to a difficult-to-control caustic burn. Topical insulin has been effective for the treatment of persistent epithelial defects. The intermediate action and low concentrations showed a shorter resolution time in neurotrophic ulcers and induced during vitreoretinal surgery (AU)

Effectiveness of topical insulin for the treatment of surface corneal pathologies.

The Purpose is to identify, through a systematic literature review, the current evidence regarding the effectiveness of topical insulin treatment in ocular surface pathologies. A literature search was implemented in Medline (Pubmed), Embase and Web Of Science medical indexing databases by using keywords such as "insulin" AND "cornea" OR "corneal" OR "dry eye" in published papers in English or Spanish within the last eleven years (2011-2022). Nine papers were identified with 180 participants from the United States, Spain, Ireland, Canada, Portugal and Malaysia, with persistent refractory epithelial defects and secondary to vitrectomy, whose extension of the lesion was from 3,75mm2 to 65.47mm2. The preparation was dissolved with artificial tears and the insulin concentration ranged from 1 IU/ml to 100 IU/ml. In all cases, the resolution of the clinical picture was complete with a healing time from 2.5 days to 60.9 days, the latter being a secondary case to a difficult-to-control caustic burn. Topical insulin has been effective for the treatment of persistent epithelial defects. The intermediate action and low concentrations showed a shorter resolution time in neurotrophic ulcers and induced during vitreoretinal surgery.

Topical management of acne scars: The uncharted terrain.

INTRODUCTION: Scarring is a common but difficult to manage consequence of acne vulgaris. The intricate balance between the degradation of collagen and its inhibition is disturbed during the formation of acne scars. We mostly rely on invasive, non-topical modalities for the treatment of acne scars which may not be indicated in all patients. There is also a need for maintainence therapies after these procedures. REVIEW: The topical agents can be utilized as individual therapy, in combination with other modalities or delivered through assisted technology like iontophoresis. Retinoids have long been tried to prevent and treat acne scars. Tacrolimus and glycolic acid are among the newer sole agents that have been explored. Ablative lasers like Er:YAG, CO2 and Microneedling are being used in combination with topical agents like silicone gel, plasma gel, lyophilized growth factors, platelet rich plasma, insulin, and mesenchymal stem cells. These procedures not only increase the permeability of the topical agents but also concomitantly improve acne scars. Iontophoresis has proven beneficial in increasing the delivery of topical estriol and tretinoin. CONCLUSION: There is lack of evidence to support the widespread use of these topical agents, and therefore, there is need for further well designed studies.

Topical Intranasal Insulin Enhances Healing of Nasal Mucosa: An Experimental Animal Study.

OBJECTIVE: Aim of this study was to evaluate the effect of topical intranasal insulin on healing of nasal mucosa in a rat model. METHODS: Forty-eight Wistar rats, weighing between 250 and 300 g and aged 10-12 weeks were used and randomized into two equal groups. 1.9 mm curette was introduced through the left nostril and 1.9 mm mucosa from the left nasal septum was curetted. Postoperatively, animals in the control group received 1 mL of physiologic saline, 3 times a day in a nasal irrigation fashion. Animals in the experimental group received 1 mL of 5 IU/mL regular insulin in saline solution. Subjects were sacrificed after 5, 10, and 15 days and macroscopic and histomorphometric evaluations were performed. RESULTS: There were no mucosal synechiae and septal perforation macroscopically. Histological examination revealed that the defect size reduction was 21% in the saline group versus 56% in the insulin group on the fifth day (p = 0.006). There was 62% defect reduction in the saline group versus 79% in the insulin group on the 10th day (p = 0.034). On the 15th day, only 67% of saline group animals had complete defect closure, whereas 100% of animals treated with insulin had complete closure (92% vs 100% mucosal defect reduction, p = 0.036). Both edema and inflammation were less in the insulin group on 15th day (p = 0.006; p = 0.023, respectively). CONCLUSION: The results from this study support the safety and efficacy of topical insulin on wound healing in the literature. This study could guide further experimental studies that examine human sinonasal wound healing.

Topical Insulin in Neurotrophic Keratopathy: A Review of Current Understanding of the Mechanism of Action and Therapeutic Approach.

Neurotrophic keratopathy is a corneal disease characterized by impaired corneal innervation. It can lead to corneal epithelial defects, ulcerations, and perforations. Topical insulin has been shown to be effective in treating this disorder. Insulin is a growth factor that can promote corneal epithelial cell proliferation and migration. In addition, it can also inhibit corneal epithelial cell apoptosis. Topical insulin has previously been found to enhance corneal wound healing. This article reviews the current understanding of the mechanism of action of topical insulin in the treatment of neurotrophic keratopathy.

Effects of topical insulin on wound healing: a meta-analysis of animal and clinical studies.

Various researches have reported that the application of topical insulin improves wound healing. Considering the lack of a quantitative comprehensive research on this matter, we conducted a meta-analysis of clinical research and experimental animal studies. Prospective and randomized controlled trials of PubMed, Embase, and Cochrane Library were conducted using appropriate search strategies to compare the effectiveness of topical application of saline and insulin on wounds. The standardized mean difference was calculated as follows: wound healing time, wound healing rate, wound area, and the percentage of wound contraction. Each study used the Cochrane risk-of-bias tool and RevMan 5.3 software to create aggregated assessments and forest plots. The quality of evidence was evaluated in accordance with the methods of the Grading of Recommendations, Assessment, Development and Evaluation working group. Four clinical and nine animal studies eligible for inclusion were included in the meta-analysis. The assessments for clinical studies were as follows: wound healing time, -2.48 [-3.44, -1.51] and wound healing rate, 22.23 [18.17, 26.28]. Meanwhile, for animal studies, the following assessments were noted: wound healing time, -1.27 [-1.75, -0.79]; wound contraction rate, 15.91 [13.88, 17.95]; and wound area, -19.3 [-21.16, -17.44]. For the measurement of the following results, only one animal study was performed, pericyte recruitment of microvessels. Based on the analysis, it can be preliminarily judged that application of topical insulin can aid wound healing.

[New progress of IGF-1 and allosteroid injection in the treatment of sudden deafness complicated with type 2 diabetes].

Glucocorticoids are a recognized treatment for sudden deafness, and there has always been a contradiction between the control of blood glucose levels and the use of glucocorticoids. The systemic use of hormones may lead to a series of adverse events, which are dose-dependent. High doses can induce an increase in blood sugar, especially for patients with type 2 diabetes, which can aggravate their condition or cause complications. The systemic application of glucocorticoids has been largely replaced by local glucocorticoids treatment. Topical insulin-like growth factor （IGF-1） is used without increasing blood sugar, thus avoiding the possible complications. The author intends to compare the local IGF-1 treatment and local glucocorticoid treatment to systemic therapy. The efficacy of local IGF-1 therapy in treating corticosteroid-refractory sudden sensorineural hearing loss combined with type 2 diabetes is reviewed.

The efficacy of topical insulin application on rat model with burn wounds treated with adipose-derived stem cells.

Overall, major burn wounds may require special care and long-term hospitalization as they not only bring complications from the wound itself, but may also compromise the immune system, or even other organs. Previous studies have indicated that topical insulin cream shortened wound closure time in second-degree burns in rats. Transplanted adipose-derived stem cells (AD-MSCs) have been developed as an alternative to treat burns and to accelerate the healing process. The aim of the present study is to investigate the effect of topical insulin gel, associated with AD-MSCs intradermal administration to heal second-degree burn wounds in rat models who were subjected to second-degree dorsal burns. The models were divided into four groups (n = 10 per group): placebo gel (C), topical insulin gel (TI), topical insulin gel and adipose-derived mesenchymal stem cells (TIMSCs) and placebo gel and adipose-derived mesenchymal stem cells (CMSCs). Wounds were assessed on a daily basis and histological evaluations were made on 5 animals from each group on the seventh and fourteenth day. There was a significant macroscopic decrease in burn wound areas in the Control (P = 0.0083), TIMSCs and CMSCs (P = 0.042) groups between the seventh and fourteenth days. The TI treatment did not show any significant change (P > 0.05) throughout this same period. The histological analysis showed significant granulation tissue formation in CMSCs and TIMSCs (P = 0.02235) treatments during the experimental period. According to the results, intradermal administration of allogenic AD-MSCs in experimental second-degree burns for short periods of time in the rat model has contributed to reducing the inflammatory phase duration, improving wound re-epithelialization, tissue granulation and wound contraction, as well as increasing collagen deposition.

Randomized Controlled Trial of Topical Insulin for Healing Corneal Epithelial Defects Induced During Vitreoretinal Surgery in Diabetics.

PURPOSE: To determine the effect of topical insulin of 3 concentrations [0.5, 1, and 2 units per drop 4 times per day (QID)] on postoperative corneal epithelial wound healing in diabetic patients. DESIGN: A double blind randomized controlled hospital-based study involving diabetic patients with postoperative corneal epithelial defect after vitreoretinal surgery. METHODS: Diabetic patients were randomized to 3 different concentrations of topical insulin (DTI 0.5, DTI 1, and DTI 2) or placebo in the control group (DNS). Primary outcome measure was the rate of corneal epithelial wound healing (mm² per hour) over pre-set interval and time from baseline to minimum size of epithelial defect on fluorescein stained anterior segment digital camera photography. Secondary outcome measure was any adverse effect of topical insulin. Follow-up was 1 month. RESULTS: Thirty-two eyes of 32 patients undergoing intraoperative corneal debridement with resultant epithelial defect (8 eyes per group) were analyzed. DTI 0.5 was superior to other concentrations achieving 100% healing rate within 72 hours of treatment compared with 62.5% in DNS, 75% in DTI 1, and 62.5% in DTI 2. Statistically, DTI 0.5 achieved significant results (P = 0.036) compared with the diabetic control group (DNS) in terms of mean rate of corneal epithelial wound healing from maximum to minimum defect size. No adverse effect of topical insulin was reported. CONCLUSIONS: Topical insulin 0.5 units QID is most effective for healing corneal epithelial defect in diabetic patients after vitrectomy surgery compared with placebo and higher concentrations. Topical insulin is safe for human ocular usage.

Effects of topical insulin on second-intention wound healing in the red-eared slider turtle (Trachemys scripta elegans) - a controlled study.

BACKGROUND: Compared with mammals, wound healing in reptiles is characterized by reduced wound contraction and longer healing times. The aim of this study is to describe the clinical and histopathological effects of topical insulin on second-intention healing of experimentally induced wounds in skin without dermal bony plates of Trachemys scripta elegans exposed to daily variations in ambient temperature and in an aquatic environment. Forty-four healthy adult females were assigned to two groups: Group 1 (n = 24) was used to assess clinical features such as wound contraction; Group 2 (n = 20) was used for histological evaluation and morphometric analysis. Topical porcine insulin (5 IU/ml diluted in glycerol) was applied daily 1 week. For each control time (2, 7, 14, 21 and 28 days post-wounding), re-epithelisation and wound remodelling were evaluated histologically and the number of main inflammatory cells (heterophils, macrophages, lymphocytes and fibroblasts) was scored. RESULTS: Mean wound contraction was higher in the insulin-treated group at each time point and differences were significant at day 28 (P < 0.0001). Histologically, these clinical findings were associated with better re-epithelisation, inflammatory response, collagen synthesis and remodelling of the wounds. Morphometrically, insulin-treated wounds had significantly higher mean counts of heterophils (day 7), macrophages (days 2, 7 and 14) and fibroblasts (days 14 and 21), whereas lymphocyte counts were significantly lower at day 21. These results demonstrate that topical insulin modifies the inflammatory response of turtle skin up-regulating inflammatory cells at early stages and promoting wound healing. CONCLUSIONS: Topical insulin is a potentially useful therapy in skin wounds of Trachemys scripta and should be evaluated in non-experimental wounds of turtles and other reptiles.

Efeito da estimulação elétrica de alta voltagem e insulina tópica em lesão cutânea experimental / Resultados de la estimulación eléctrica por alta voltaje e insulina tópica en lesión cutánea experimental / Effect of high-voltage electrical stimulation and topical insulin on experimental cutaneous lesions

RESUMO O objetivo deste estudo foi avaliar o efeito da estimulação elétrica de alta voltagem (EEAV) catódica, associada à insulina tópica, em lesão tegumentar de ratos. Para isso, foram utilizados 42 ratos Wistar (240±30 g), submetidos a retirada cirúrgica de 1 cm2 de pele do dorso em seis grupos (n=7), tratados por sete dias consecutivos: controle (C), estimulação elétrica placebo (EP), estimulação elétrica catódica (EE), insulina tópica (IT), insulina placebo (IP) e EEAV associada a insulina tópica (EE+I). A EEAV foi administrada 24 horas após a cirurgia, 30 minutos por dia, com frequência de 100 Hz e voltagem média de 60 V, mantida no limiar motor. Áreas das lesões foram registradas macroscopicamente no primeiro, quarto e oitavo dia, sendo submetidas a tratamento histológico para inclusão em paraplast® e coloração em hematoxilina e eosina. A epitelização e o perfil numérico das células foram obtidos por análises histométricas. Utilizou-se o teste de Shapiro-Wilk e ANOVA one-way seguida de Bonferrone (p<0,05). Observou-se redução significativa na área da lesão no oitavo dia de tratamento, nos grupos EE e EE+I em relação aos demais grupos. A reepitelização não diferiu entre os grupos, mas a distância entre as bordas da lesão foi menor nos grupos EE e EE+I. Nesses grupos houve aumento significativo (p<0,05) no número de fibroblastos e diminuição de leucócitos. Pode-se concluir que a EEAV catódica acelerou o processo de reparação da lesão, não demonstrando efeito adicional com a aplicação da insulina tópica.

RESUMEN El propósito de este estudio fue evaluar el resultado de la estimulación eléctrica por alta voltaje (EEAV) catódica, asociada a la insulina tópica, en lesión cutánea de ratas. Para ello, se utilizaron 42 ratas Wistar (240±30 g), les sometieron a cirugía de retirada de 1 cm2 de piel del dorso en 6 grupos (n=7), y les trataron por siete días consecutivos: control (C), estimulación eléctrica placebo (EP), estimulación eléctrica catódica (EE), insulina tópica (IT), insulina placebo (IP) y EEAV asociada a la insulina tópica (EE+I). Se aplicó la EEAV 24 horas después de la cirugía, 30 minutos por día, con frecuencia de 100 Hz y voltaje de media tensión de 60 V, y la mantuvo en el umbral motor. Se registraron las áreas de las lesiones macroscópicamente en el primer, cuarto y octavo día, y las sometieron al tratamiento histológico para inclusión en paraplast® y tinción hematoxilina-eosina. Se obtuvo la epitelización y el perfil numérico de las células por análisis histométricos. Se empleó la prueba Shapiro-Wilk y ANOVA one way de Bonferroni (p<0,05). Se redujo significativamente el área de la lesión en el octavo día del tratamiento en los grupos EE y EE+I comparados a los demás grupos. La reepitalización no fue distinta entre los grupos, sin embargo, la distancia entre los bordes de la lesión fue menor en los grupos EE y EE+I. También en estos grupos aumentó significativamente (p<0,05) el número de fibroblastos y disminuyeron los leucocitos. Se concluye que la EEAV catódica aceleró el proceso de reparación de la lesión, pero no ocurrió resultado con la aplicación de la insulina tópica.

ABSTRACT This study aims to evaluate the effect of cathodic high voltage electrical stimulation (HVES), associated with topical insulin, on rat integumentary lesions. For this purpose, 42 Wistar rats (240±30 g) were submitted to surgical removal of 1 cm2 of dorsal skin and divided into six groups (n=7), treated for seven consecutive days: Control (C), placebo electrical stimulation (PES), cathodic electrical stimulation (ES), topical insulin (TI), placebo insulin (PI) and HVES associated with topical insulin (ES+I). HVES was administered 24 hours after surgery, 30 minutes per day, with a frequency of 100 Hz and a mean voltage of 60 V, maintained at the motor threshold. Lesion areas were recorded macroscopically on the first, fourth and eighth day, submitted to histological treatment for inclusion in paraplast® and staining in Hematoxylin and Eosin. Epithelialization and the numerical profile of the cells were obtained by histometric analysis. The Shapiro-Wilk and Anova one-way test was followed by the Bonferroni (p<0.05). There was a significant reduction in the area of the lesion by the eighth day of treatment, both in the ES and ES+I groups, when compared with other groups. Reepithelialization did not differ between groups, but the distance between the edges of the lesion was lower in the ES and ES+I groups. These same groups showed a significant increase (p<0.05) in the number of fibroblasts and a decrease in leukocytes. Thus, we can conclude that cathodic HVES accelerated the lesion repair process, with the topical application of insulin showing no additional effect.