Norma Roda

Effect of oxiplex* films (PEO/CMC) on adhesion formation and reformation in rabbit models and on peritoneal infection in a rat model

OBJECTIVE To assess the efficacy of Oxiplex (FzioMed, Inc., San Luis Obispo, CA) barriers. DESIGN Film of polyethylene oxide and carboxymethylcellulose (Oxiplex) were tested for strength and tissue adherence. Films were selected for evaluation in models for biocompatability and adherence. Three films were selected for evaluation in efficacy studies, and one was evaluated for effects on bacterial peritonitis. Handling characteristics of Oxiplex film were evaluated via laparoscopy. SETTING University laboratory. PATIENT(S) Rabbits, rats, pigs. INTERVENTION(S) Placement of Oxiplex prototypes at the site of injury. MAIN OUTCOME MEASURE(S) Mechanical properties, biocompatibility, tissue adherence, adhesion development, infection potentiation, and device handling. RESULT(S) Mechanical tests indicated that tensile strength and elongation were inversely correlated. All films tested had excellent tissue adherence properties. Selected films, based on residence time and biocompatibility, prevented adhesion formation in all animals and were highly efficacious in preventing adhesion reformation. The optimal Oxiplex prototype prevented adhesion reformation in 91% of the animals. This Oxiplex film, dyed to allow visualization, prevented adhesion reformation and did not affect bacterial peritonitis. In a laparoscopic model, the Oxiplex film, delivered in FilmSert forceps, via a 5.0-mm trocar, rapidly unfurled and could be easily applied to tissue with strong adherence. CONCLUSION(S) These data show development of an adhesion prevention material that is tissue adherent, can be placed via laparoscopy, and does not affect host resistance.

Development of angiotensin (1-7) as an agent to accelerate dermal repair

Angiotensin II has been shown to be a potent agent in the acceleration of wound repair. Angiotensin (1‐7), a fragment of angiotensin II that is not hypertensive, was found to be comparable to angiotensin II in accelerating dermal healing. This activity was evaluated in four models: rat and diabetic mouse full‐thickness excisional wounds; rat random flap; and guinea pig partial thickness thermal injury. In all models, angiotensin (1‐7) was comparable to angiotensin II. Angiotensin (1‐7) accelerated the closure of wounds in diabetic mice and rats. In diabetic mice the resultant tissue at day 25 after injury was more comparable to normal tissue than the fibrotic scar observed in placebo‐treated wounds. In the random flap model, angiotensin (1‐7) was comparable to angiotensin II in maintaining flap viability (approximately 82%) and flap survival (40%). Finally, angiotensin (1‐7) increased proliferation in the hair follicles at the edge of the wound and site of thermal injury, and the number of patent blood vessels on day 7 after partial thickness thermal injury. These data may be partially explained by the effect of angiotensin II and angiotensin (1‐7) on keratinocyte proliferation. While platelet‐derived growth factor had no effect on keratinocyte proliferation, angiotensin II and angiotensin (1‐7) significantly increased keratinocyte proliferation. These data show that angiotensin(1‐7) is comparable to angiotensin II in accelerating skin repair. Furthermore, the hypertensive and wound healing effects can be separated within the family of angiotensin peptides.

Histological evaluation of the effects of angiotensin peptides on wound repair in diabetic mice

Abstract:  Recent studies have shown that angiotensin peptides accelerate dermal repair. Histological observation of samples taken at the termination of studies showed that the wounds treated with peptides were more mature and organized by day 25 after full thickness excision in diabetic mice. However, the mechanism by which this acceleration occurs has not been determined. In the experiments described here, the effect of angiotensin peptides [AII, A(1–7) and NorLeu3‐A(1–7)] on the quality of the healing wound was evaluated histologically. Administration of the peptides accelerated collagen deposition, re‐epithelialization and new blood vessel formation. By day 4, the percentage of the wound with collagen increased two‐ to six‐fold depending upon the peptide. The increase by angiotensin peptides continued throughout the experimental period. On days 4 and 7 (only) after injury, exposure to angiotensin peptides increased the number of blood vessels at the wound site two‐ to three‐fold. Finally, the percentage of the wound site covered with new epithelium increased after administration of angiotensin peptides. Re‐epithelialization was observed as early as day 4 in wounds treated with angiotensin peptides. The increase was greater at later time points [up to 8‐fold at day 14 with NorLeu3‐A(1–7)]. Fibroblast infiltration and proliferation occurred earlier in wounds treated with angiotensin peptides. Wounds treated with A(1–7) and NorLeu3‐A(1–7) had an increase in neutrophils and macrophages on day 4 after wounding. Overall, administration of these peptides resulted in a healing site that was more mature, including reorganization of the collagen into a basket‐weave appearance. Further, these studies confirm the superiority of NorLeu3‐A(1–7) to AII and A(1–7) in wound healing evaluated at a microscopic level.

Acceleration of healing, reduction of fibrotic scar, and normalization of tissue architecture by an angiotensin analogue, NorLeu3-A(1-7).

Angiotensin peptides have been demonstrated to modulate cellular proliferation, angiogenesis, and dermal repair. In this report, the effects of an analogue of the active angiotensin peptide angiotensin(1-7), namely norLeu3-angiotensin(1-7) (NorLeu3-A(1-7)), on the healing of epithelial wounds are presented. Three models were used to evaluate the normal (rats) and delayed (diabetic mice) healing responses of full-thickness excision wounds and the healing responses of full-thickness incision wounds (rats). NorLeu3-A(1-7) was superior to the naturally occurring angiotensin peptide angiotensin(1-7) and to Regranex (Ortho McNeil, Somerville, N.J.) (a formulation of recombinant platelet-derived growth factor used clinically for the treatment of diabetic ulcers) in accelerating tissue repair. By day 9 (normal rats) and day 11 (diabetic mice), the differences in the rates of closure of full-thickness excision wounds between NorLeu3-A(1-7) and Regranex were statistically significant (n = 5 per group). Full healing was observed for 60 percent of the diabetic mice treated topically with NorLeu3-A(1-7) by day 18 after injury, at which time full healing of wounds on placebo-treated or Regranex-treated diabetic mice was not observed. In the rat incision model, accelerated healing and reduced gross appearance of scarification were observed. Administration of NorLeu3-A(1-7) reduced fibrosis and scarring in the healing wounds. This action was more pronounced with longer administration of the peptide after injury. In fact, if systemic administration of the peptide (NorLeu3-A(1-7)) was continued during the remodeling phase, then the formation of new adnexal structures at the center of full-thickness excision wounds was observed, with an increase in the appearance of small immature hair follicles at the sites of the excision wounds. The action of this peptide was blocked by the AT receptor antagonist d-Ala7-angiotensin(1-7), which suggests that this receptor is involved in the healing responses to exogenous NorLeu3-A(1-7). These data suggest that this novel angiotensin peptide has the potential to be of benefit in accelerating wound repair and reducing scar formation.

Angiotensin II improves random-flap viability in a rat model.

Angiotensin II (AII) is a naturally occurring peptide that has been shown to be angiogenic, cause the proliferation of several primary cell types (including endothelial cells), accelerate the repair of dermal injuries, and increase production of growth factors and extracellular matrix. The effect of a single administration of AII on the viability and vascularity of a random flap was assessed in a rat model. In the control model, the viability of the distal portion of the flap was reduced consistently by postoperative day 8. Initially, AII was administered in an aqueous vehicle (phosphate-buffered saline [PBS]) and a viscous vehicle (10% carboxymethyl cellulose [CMC]). Administration of 1 mg per milliliter AII in PBS did not affect the viability of random flaps (1.2 x 7 cm) in this animal model. However, a single administration of a higher dose of AII in PBS (10 mg per milliliter) or 1 mg per milliliter AII in the CMC vehicle resulted in 67% of the grafts being fully viable at postsurgical day 12, in contrast to vehicle-treated control flaps, none of which were fully viable at day 12. Furthermore, the portion of the flap that was viable was increased significantly (p < or = 0.05). Subsequently, a study was conducted to assess the dose-response curve for AII in a CMC vehicle in this rat model. As the dose of AII was reduced, the percentage of animals with fully viable flaps and the percentage of the flap that was viable decreased correspondingly. Administration of 0.03 mg per milliliter AII and greater increased significantly (p < or = 0.05) the viability of the flaps. In conclusion, AII appears to be highly efficacious in increasing the percentage of distal flap surface area survival when administered as a single topical dose to the wound bed.

Angiotensin-(1–7) Administration Reduces Oxidative Stress in Diabetic Bone Marrow

Diabetics have an increased risk of developing cardiovascular disease, in part due to oxidative stress, resulting in endothelial nitric oxide synthase (eNOS) dysfunction. Studies have demonstrated that angiotensin-(1-7) [Ang-(1-7)] can activate eNOS activity. Because the bone marrow is a primary source of a number of progenitors important in physiological homeostasis and healing, the goal of this study was to evaluate the in vivo effects of Ang-(1-7) treatment on oxidative stress and the ensuing nitrative stress in diabetic bone marrow and its potential pathways. BKS.Cg-Dock7(m) +/+ Lepr(db)/J mice and their heterozygous controls were administered Ang-(1-7) alone or combined with A-779, losartan, PD123,319, nitro-l-arginine methyl ester, or icatibant sc for 14 d. The bone marrow was then collected to measure nitric oxide levels, eNOS phosphorylation, and expression of nitric oxide synthase, superoxide dismutase, and p22-phox. Nitric oxide levels in the bone marrow were significantly decreased in diabetic mice, and Ang-(1-7) treatment was able to significantly increase these measures (P < 0.01). This effect was blocked by the coadministration of PD123,319, A-779, nitro-l-arginine methyl ester, and icatibant. In addition, Ang-(1-7) treatment reversed the paradoxical increase in eNOS and neuronal nitric oxide synthase expression and decreased the phosphorylation of eNOS at Thr495 seen in diabetic mice. Ang-(1-7) also reversed diabetes-induced production of reactive oxygen species by decreasing p22-phox expression and increasing superoxide dismutase 3 expression, leading to a significant reduction in 3-nitrotyrosine formation in diabetic bone marrow (P < 0.05). Our findings demonstrate that Ang-(1-7) administration decreases diabetes-induced oxidative stress in the bone marrow and modifies pathways involved in eNOS dysfunction.

Effect of NorLeu3-A(1-7) on scar formation over time after full-thickness incision injury in the rat

Previous studies have shown that the angiotensin peptide NorLeu3‐A(1‐7) accelerates dermal healing and reduces scar formation. In this report, the effect of this peptide on scar formation is more fully delineated. The effect of surgical day, time after injury, and observer on the clinical appearance of the incision were determined. Clinical observations of incision site, including inflammation, opening of the injury, and appearance of scar, were conducted by two observers blinded to treatment (two observations per time point) twice weekly. Opening of the incision occurred in 35–40% of incisions early (days 4 and 7) after injury. Administration of NorLeu3‐A(1‐7) at the time of injury reduced the incidence of opening at day 7 to approximately 20%. Further, the length of the wound opening was significantly reduced in the peptide‐treated incisions at day 7. Starting on day 14 after injury, scar formation was evaluated. Up to 80–90% of control animals had observable scars starting on day 14. Thereafter, the scar remodeled with fewer incisions having visible scar on day 28. With administration of NorLeu3‐A(1‐7), significantly fewer incisions had observable scars starting on day 14 and throughout the study. As few as 20% of the incisions had observable scars on day 28. The histological appearance of the healing wound was also evaluated at weekly intervals starting on day 7 and continued until day 42. At day 7, the maximal number of fibroblasts at the wound site was observed. Thereafter, the number gradually reduced, reaching a plateau at day 28. The administration of peptide had no effect on fibroblast number at the incision site. A similar pattern was observed in the thickness of the epidermis with the resolution of the hyperplastic phase at day 21. Administration of the peptide significantly increased epidermal height at day 7. Blood vessel formation peaked on day 21 and 28 in control wounds and was further enhanced by peptide administration during the neovascularization phase. After day 28, blood vessel number was comparable between control and treated incisions. Collagen deposition and remodeling were increased by the administration of NorLeu3‐A(1‐7) at the time of injury.

Effects of Combined Radiation and Burn injury on the Renin-Angiotensin System

The renin–angiotensin system (RAS) plays an important role in wound repair; however, little is known pertaining to RAS expression in response to thermal injury and the combination of radiation plus burn injury (CRBI). The purpose of this study was to test the hypothesis that thermal injury modifies expression of RAS components and CRBI delayed this up‐regulation of RAS. Skin from uninjured mice was compared with mice receiving local thermal injury or CRBI (injury site). Skin was analyzed for gene and protein expression of RAS components. There was an initial increase in the expression of various components of RAS following thermal injury. However, in the higher CRBI group there is an initial decrease in AT1b (vasoconstriction, pro‐proliferative), AT2 (vasodilation, differentiation), and Mas (vasodilation, anti‐inflammatory) gene expression. This corresponded with a delay and decrease in AT1, AT2, and MAS protein expression in fibroblasts and keratinocytes. The reduction in RAS receptor positive fibroblasts and keratinocytes correlated with a reduction in collagen deposition and keratinocyte infiltration into the wounded area resulting in a delay of reepithelialization following CRBI. These data support the hypothesis that delayed wound healing observed in subjects following radiation exposure may be in part due to decreased expression of RAS.

Accelerated hematopoietic recovery with angiotensin-(1–7) after total body radiation

Purpose: Angiotensin (1–7) [A(1–7)] is a component of the renin angiotensin system (RAS) that stimulates hematopoietic recovery after myelosuppression. In a Phase I/IIa clinical trial, thrombocytopenia after chemotherapy was reduced by A(1–7). In this study, the ability of A(1–7) to improve recovery after total body irradiation (TBI) is shown with specific attention to radiation-induced hematopoietic injury. Materials and methods: Mice were exposed to TBI (doses of 2–7 Gray [Gy]) of cesium 137 gamma rays, followed by treatment with A(1–7), typical doses were 100–1000 μg/kg given once or once daily for a specified number of days depending on the study. Animals are injected subcutaneously via the nape of the neck with 0.1 ml drug in saline. The recovery of blood and bone marrow cells was determined. Effects of TBI and A(1–7) on survival and bleeding time was also evaluated. Results: Daily administration of A(1–7) after radiation exposure improved survival (from 60% to 92–97%) and reduced bleeding time at day 30 after TBI. Further, A(1–7) increased early mixed progenitors (3- to 5-fold), megakaryocyte (2- to 3-fold), myeloid (3- to 6-fold) and erythroid (2- to 5-fold) progenitors in the bone marrow and reduced radiation-induced thrombocytopenia (RIT) (up to 2-fold). Reduction in the number of treatments to 3 per week also improved bone marrow recovery and reduced RIT. As emergency responder and healthcare systems in case of nuclear accident or/and terrorist attack may be overwhelmed, the consequence of delayed initiation of treatment was ascertained. Treatment with A(1–7) can be delayed up to 5 days and still be effective in the reduction of RIT or acceleration of bone marrow recovery. Conclusions: The data presented in this paper indicate that A(1–7) reduces the consequences of critical radiation exposure and can be initiated well after initial exposure with maximal effects on early responding hematopoietic progenitors when treatment is initiated 2 days after exposure and 5 days after exposure for the later responding progenitors and reduced thrombocytopenia. There was some effect of A(1–7) even when given days after radiation exposure.

A new model for experimental tendon adhesions in the chicken

&NA; A minimally invasive model using a manual abrader to induce adhesions in the chickens central digit is described. The flexor synovial sheath and the profundus tendon were abraded with access through small flaps at the level of the proximal and distal phalanges of the avian long toes. The birds were divided into two groups according to the severity of the induced trauma. Group I birds received an abrasion injury and were euthanized to allow biomechanical testing 5 weeks postoperatively. Group II birds had a more severe abrasion and were euthanized similarly and tested 5 weeks after surgery. Results were compared with nonsurgical controls. Long toe function was evaluated weekly by measuring (1) the range of active flexion of each interphalangeal joint, resolved to total angular range; (2) the grasping ability on graded‐diameter perches; and (3) the flexion deficit of the long toe. Postmortem biomechanical properties of the adhesions were measured. There was a significant difference between the unoperated controls and abraded digits of both groups in all parameters (p < 0.001). There was, in addition, a marked change in most of the measured parameters between groups I and II. In group I digits the functional and biomechanical deficit was less than group II. In summary, this animal model of long‐segment abrasive injury to the tendon and sheath is a simple and reproducible method to generate adhesions and can be used for the evaluation of treatment modalities for adhesion prevention. Jaibaji M, Brody GS, Rodgers K, Espinoza T, Roda N, Maldonado S, Pines E, diZerega G. A new model for experimental tendon adhesions in the chicken. Ann Plast Surg 2000;44:205‐210