Development of a guinea pig cutaneous radiation injury model using low penetrating X-rays.

PURPOSE: A guinea pig skin model was developed to determine the dose-dependent response to soft X-ray radiation into the dermis. MATERIALS AND METHODS: X-ray exposure (50 kVp) was defined to a 4.0 × 4.0 cm area on the lateral surface of a guinea pig using lead shielding. Guinea pigs were exposed to a single fraction of X-ray irradiation ranging from 25-79 Gy via an XRAD320ix Biological Irradiator with the collimator removed. Gross skin changes were measured using clinical assessments defined by the Kumar scale. Skin contracture was assessed, as well as histological evaluations. RESULTS: Loss of dermal integrity was shown after a single dose of soft X-ray radiation at or above 32 Gy with the central 2.0 × 2.0 cm of the exposed site being the most affected. Hallmarks of the skin injury included moist desquamation, ulceration and wound contracture, as well as alterations in epithelium, dermis, muscle and adipose. Changes in the skin were time- and radiation dose-dependent. Full-thickness injury occurred without animal mortality or gross changes in the underlying organs. CONCLUSIONS: The guinea pig is an appropriate small animal model for the short-term screening of countermeasures for cutaneous radiation injury (CRI).

Effect of combined radiation injury on cell death and inflammation in skin.

In the event of a nuclear disaster, the individuals proximal to the source of radiation will be exposed to combined radiation injury. As irradiation delays cutaneous repair, the purpose of this study was to elucidate the effect of combined radiation and burn injury (CRBI) on apoptosis and inflammation at the site of skin injury. Male C57Bl/6 mice were exposed to no injury, thermal injury only, radiation only (1 and 6 Gy) and CRBI (1 and 6 Gy) and euthanized at various times after for skin collection. TUNEL staining revealed that the CRBI 6 Gy group had a delayed and increased apoptotic response. This correlated with decreased recovery of live cells as compared to the other injuries. Similar response was observed when cleaved-caspase-3 immunohistochemical staining was compared between CRBI 6 Gy and thermal injury. TNFR1, caspase 8, Bax and IL-6 mRNA expression revealed that the higher CRBI group had delayed increase in mRNA expression as compared to thermal injury alone. RIPK1 mRNA expression and necrotic cell counts were delayed in the CRBI 6 Gy group to day 5. TNF-α and NFκB expression peaked in the CRBI 6 Gy group at day 1 and was much higher than the other injuries. Also, inflammatory cell counts in the CRBI 6 Gy group were lower at early time points as compared to thermal injury by itself. These data suggest that CRBI delays and exacerbates apoptosis and inflammation in skin as well as increases necrosis thus resulting in delayed wound healing.

Angiotensin-(1-7) synergizes with colony-stimulating factors in hematopoietic recovery.

PURPOSE: Angiotensin (1-7) [A(1-7)] is a bioactive peptide of the renin angiotensin system that stimulates the number of bone marrow progenitors and hematopoietic recovery after myelosuppression. We evaluated the combination of A(1-7) with colony-stimulating factors, Neupogen and Epogen, on bone marrow progenitors and the recovery of circulating formed elements following chemotherapy. METHODS: Mice were injected with gemcitabine followed 2 days later with A(1-7). Circulating blood cells and bone marrow progenitors were measured over time. RESULTS: Combination of A(1-7) with Neupogen (the latter given only 3 days starting at the white blood cell nadir) decreased the amount of Neupogen needed for optimal recovery by 10-fold. The progenitors measured include CFU-GEMM, CFU-GM, CFU-Meg and BFU-E. A(1-7) increased recovery of all progenitors when given alone or in combination with Neupogen above that with Neupogen alone. Combination of A(1-7) with Epogen slightly increased (not significantly) red blood cell concentrations above those achieved by Epogen alone. However, in this model, A(1-7) or A(1-7) in combination with Epogen increased all erythroid progenitors with the largest effect on early erythroid progenitors (immature BFU-E). CONCLUSIONS: Neupogen and Epogen acted synergistically with A(1-7) to increase the concentration of myeloid, megakaryocytic and erythroid progenitor cells in the bone marrow following chemotherapy suggesting that A(1-7)'s multilineage effect on early progenitors in the marrow facilitates proliferation in response to lineage-specific growth factors.

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 AT(1b) (vasoconstriction, pro-proliferative), AT(2) (vasodilation, differentiation), and Mas (vasodilation, anti-inflammatory) gene expression. This corresponded with a delay and decrease in AT(1) , AT(2) , 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.

Expression of intracellular filament, collagen, and collagenase genes in diabetic and normal skin after injury.

Reports have shown differences in gene expression in the skin of diabetic and normal mice both at baseline and after injury. Cluster analysis identified distinct expression patterns within intermediate filaments and extracellular proteins. This report addresses the effect of diabetes and injury on the expression of keratin-associated proteins, keratin complexes, procollagen, and collagenase (matrix metalloproteinase; MMP) genes. At baseline keratin-associated proteins and keratin complexes gene expression was increased in diabetic mice. After surgery, the level of expression for keratin-associated proteins and keratin complexes genes decreased in diabetic mice, but did not change in normal mice. If the expression of a procollagen gene differed between diabetic and normal mice, the expression was lower in diabetic mice. Procollagen gene expression was elevated after skin excision compared with noninjured skin. At baseline, the level of MMP and tissue inhibitor of metalloproteinase gene expression was comparable between mouse strains. With injury, the expression of several MMP genes was increased in both mouse strains, but to higher levels in diabetic mice. At day 7, the level of MMP-9 activity in granulation tissue was elevated. This alteration may contribute to delayed healing in diabetic mice. Therefore, differences in gene expression exist between mouse strains and can assist in understanding of physiological manifestations, including delayed healing, in diabetic mice.

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.

Synergistic effects of co-administration of angiotensin 1-7 and Neupogen on hematopoietic recovery in mice.

PURPOSE: Angiotensin 1-7 [A(1-7)] is a seven amino acid peptide that has been shown to increase the proliferation of epidermal stem cells after dermal injury and the number of hematopoietic progenitors in the bone marrow of myelosuppressed mice. In this study, the effect of combining A(1-7) with Neupogen on hematopoietic recovery and bone marrow progenitors was evaluated. MATERIALS AND METHODS: Intravenous 5-fluorouracil (5FU) was administered to induce myelosuppression. Administration of A(1-7) and/or Neupogen was initiated 2 days after chemotherapy. Angiotensin II (AII) and A(1-7) binding were assessed by flow cytometric analysis. Hematopoietic progenitors were counted by colony forming assays. Recovery of formed elements in the blood was evaluated by hemocytometer. RESULTS: Flow cytometric analysis indicated that the number of early hematopoietic progenitors (Lin(-)Sca1(+)cKit(+)) that bind AII or A(1-7) increased 5-7 days after intravenous injection of 150 mg/kg 5FU. Further, administration of A(1-7) led to a slight increase in the number of circulating leukocytes and platelets after this chemotherapeutic regimen. When given in combination with a subclinical dose of Neupogen, a synergistic effect on the number of circulating leukocytes was observed, but there was no further effect on the number of circulating platelets. In myelosuppressed mice, A(1-7) had its most profound effect on the number of hematopoietic progenitors in the bone marrow. The progenitors evaluated in the study included BFU-E, CFU-Meg, CFU-GM and CFU-GEMM. There was an increase in the number of these progenitors in the bone marrow, indicating an effect on all hematopoietic lineages. When given in combination with Neupogen, these effects were synergistic for the numbers of BFU-E and CFU-Meg (Neupogen by itself had no effect) and for the myeloid progenitors at lower doses of A(1-7). CONCLUSIONS: These results suggest that these hematopoietic agents act at different sites within the hematopoietic cascade and that combining these two agents may be of benefit in the treatment of hematopoietic disorders.

Reduction of epidural fibrosis in lumbar surgery with Oxiplex adhesion barriers of carboxymethylcellulose and polyethylene oxide.

BACKGROUND CONTEXT: Postsurgical epidural adhesions and fibrosis after surgery for lumbar disc herniation are a consequence of normal wound healing. The presence of fibrosis renders reoperations risky, and in some patients fibrosis may lead to nerve root tethering. PURPOSE: One approach to minimizing the risk of developing epidural adhesions is to provide a barrier between the dural membrane and the healing connective tissues. The purpose of these studies was to evaluate such a barrier device. STUDY DESIGN/SETTING: In vivo investigation in an animal model at a university laboratory. PATIENT SAMPLE: Rabbit. OUTCOME MEASURES: Gross and histomorphic evaluation. METHODS: Barriers comprised of carboxymethylcellulose (CMC) and polyethylene oxide (PEO) (Oxiplex; FzioMed, Inc., San Luis Obispo, CA) were studied as devices to reduce epidural adhesion formation in rabbit laminotomy and laminectomy models. The barriers tested were either a gel alone (gel) or a gel covered with a film (gel/film combination). Two laminotomy or laminectomy sites (depending on the surgical method) were created in each rabbit at L4 and L6. One site was treated with a CMC/PEO gel, or CMC/PEO gel/film combination, and the other site served as a surgical control. Two surgical models that differed in the extent of adhesion formation at untreated injury sites and the method of injury generation were used. RESULTS: Model A, which did not incorporate dural abrasion, resulted in up to 40% adhesion-free laminectomy sites in controls. Model B, which did incorporate abrasion of the dural membrane, resulted in less than 10% adhesion-free laminotomy sites in controls. Compositions of CMC/PEO gels (2.5% to 10% PEO) and films (22.5% PEO) were tested in both models. Efficacy parameters included measuring the number of sites free of epidural fibrosis and reduction in the severity of fibrosis (adhesions). Both gels and gel/film combinations consistently reduced the frequency and the extent of epidural fibrosis in both models. Gels of CMC/PEO containing a higher content of PEO (10%) and a higher molecular weight of PEO (4.4 mD) were most effective in Model B and resulted in up to 84% laminotomy sites with minimal or no epidural fibrosis, whereas controls exhibited over 90% of the sites with epidural fibrosis. Histological evaluation of the surgical sites indicated that the reduction of epidural fibrosis was accompanied by normal bone healing. In addition, these experiments demonstrated that the gel/film combination provided no additional benefit to that obtained by the gel alone. CONCLUSIONS: Gels of CMC/PEO reduced epidural fibrosis and did not impair normal heal ing.

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.

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

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 mature and organized by day 25 after full thickness excision in diabetic mice. However, the mechanisms by which this acceleration occurs has not been determined. In the experiments described here, the effect of angiotensin peptides (AII, A(1-7) and NorLeu (3)-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 vessels 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 9 (only) after injury, exposure to angiotensin peptides increased the number of blood vessels at 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 ith angiotensin peptides. The increase was greater at later time points (up to 8-fold ar day 14 with NorLeu(3)-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 NorLeu(3)-A(1-7) to AII and A(1-7) in wound healing evaluated at a microscopic level.