Total Panax notoginseng saponin inhibits balloon injury-induced neointimal hyperplasia in rat carotid artery models by suppressing pERK/p38 MAPK pathways

Total Panax notoginseng saponin (TPNS) is the main bioactivity compound derived from the roots and rhizomes of Panax notoginseng (Burk.) F.H. Chen. The aim of this study was to investigate the effectiveness of TPNS in treating vascular neointimal hyperplasia in rats and its mechanisms. Male Sprague-Dawley rats were randomly divided into five groups, sham (control), injury, and low, medium, and high dose TPNS (5, 10, and 20 mg/kg). An in vivo 2F Fogarty balloon-induced carotid artery injury model was established in rats. TPNS significantly and dose-dependently reduced balloon injury-induced neointimal area (NIA) (P<0.001, for all doses) and NIA/media area (MA) (P<0.030, for all doses) in the carotid artery of rats, and PCNA expression (P<0.001, all). The mRNA expression of smooth muscle (SM) α-actin was significantly increased in all TPNS groups (P<0.005, for all doses) and the protein expression was significantly increased in the medium (P=0.006) and high dose TPNS (P=0.002) groups compared to the injury group. All the TPNS doses significantly decreased the mRNA expression of c-fos (P<0.001). The medium and high dose TPNS groups significantly suppressed the upregulation of pERK1/2 protein in the NIA (P<0.025) and MA (P<0.004). TPNS dose-dependently inhibited balloon injury-induced activation of pERK/p38MAPK signaling in the carotid artery. TPNS could be a promising agent in inhibiting cell proliferation following vascular injuries.

Chronic renal failure promotes intimal hyperplasia of arteriovenous fistula in mice / 基础医学与临床

Objective To find the effect of chronic renal failure on the development of neointimal hyperplasia and the role of monocyte chemokine-1 (MCP-1) after arteriovenous fistula in mice.Methods We created AVF (common carotid artery to jugular vein in an end-to-end anastomosis) in mice with or without chronic renal failure (renal ablation or sham operation).The outflow of AVF was harvested at 3 weeks postoperative the vascular tissue.The pathological changes were examined.The level of blood urea nitrogen (BUN) and the degree of intimal hyperplasia were analysed.The protein and mRNA expression of alpha smooth muscle actin (SMA), Ki-67,NF-κB and MCP-1 were detected by immunohistochemistry, RT-PCR and Western blot.Results 1)Compared with the control group, the blood BUN level of the experimental group was significantly higher and the intimal hyperplasia was more serious, meanwhile, the lumen was more narrow (P<0.05).2)In the experimental group, the expression of α-SMA, Ki-67, NF-κB and MCP-1 was significantly increased (P<0.05).3)MCP-1 promoted the proliferation of vascular smooth muscle cells.Conclusions Chronic renal failure promote the development of neointimal hyperplasia, which may be related to the increase of MCP-1 expression.

Administration of a High-Dose Erythropoietin-Stimulating Agent in Hemodialysis Patients is Associated with Late Arteriovenous Fistula Failure

PURPOSE: Investigating the risk of vascular access failure is critical for maintenance hemodialysis (MHD) patients. Erythropoietin stimulating agents (ESA) typically used for anemia of chronic kidney disease (CKD) may also stimulate neointimal hyperplasia, which is the most important factor in late arteriovenous fistula (AVF) failure. The aim of this study was to investigate whether ESA treatment is associated with late AVF failure. MATERIALS AND METHODS: The late AVF failure group comprised 51 patients who underwent percutaneous intervention or surgery for fistula revision after successful use for at least three months. There were 51 controls whose AVF had been patent for at least 24 months. RESULTS: The mean time from the first cannulation to late loss of AVF patency was 8.4±4.2 months. The average weekly dose of ESA was significantly higher in patients with AVF failure (4782.2±2360.5 IU/mL/wk vs. 7161.8±2775.2 IU/mL/wk, p<0.001). The only independent predictor of late AVF failure in multivariate analysis was high average ESA dose (odds ratio=1.015, 95% confidence interval=1.002–1.028, p=0.022). CONCLUSION: Patients with late AVF patency loss exhibit an association with a higher dose of ESA, although causality is unproven. Further study to elucidate potential mechanisms is warranted.

Inhibitory effects of ginsenoside Re on vascular neointimal hyperplasia induced by balloon-injury and ERK signaling in rats / 中国药学杂志

OBJECTIVE: To investigate the inhibitory effect of ginsenoside Re on vascular neointimal hyperplasia induced by balloon-injury and probe its molecular mechanism in rats. METHODS: The rat carotid artery neointimal hyperplasia model was established by rubbing the endothelia with a balloon in male Sprague-Dawley rats, then animals were intrapritoneally injected with distilled water in model group and sham operation group or with ginsenoside Re 6, 12 and 24 mg·kg-1·d-1 in other endothelia rubbed groups. After 14 consecutive days, the injuried artery was taken for H&E staining, the histopathological observation and detecting the neointimal area as well as the ratio of neointimal area/media area were taken to evaluate the vescular intimal hyperplasia level. For probing the molecular mechanism, the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) was detected at the transcript levels by real time RT-PCR, and the protein expressions of MKP-1 and phosphorylation extracellular signal-regulated kinase 1/2 (pERK12) were examined by immunohistochemistry and analyzed with Image-Pro Plus. RESULTS: Compared with the endothelia rubbing model group, ginsenoside Re 6, 12, 24 mg·kg-1·d-1 medications significantly improved the histopathological changes induced by baloon-injury, decreased the elevated neointimal area and the ratio of neointimal area/media area induced by baloon-injury; ginsenoside Re administration could also down-regulate the elevated pERK1/2 protein expression, and significantly up-regulated the decreased MPK-1 mRNA and protein expressions induced by baloon-injury. CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by baloon-injury in rats, the molecular mechanism is related to its inhibition effect on ERK signaling.

Inhibitory effect of evodiamine on neointima hyperplasia of rat carotid arteries with balloon injury / 中国药学杂志

OBJECTIVE: To examine the inhibitory effects of evodiamine on vascular neointimal hyperplasia in balloon-injured carotid artery of rats and explore the possible mechanisms. METHODS: Healthy male Sprague Dawley rat vascular neointimal hyperplasia model was made by rubbing the endothelia of common carotid artery with a balloon, and then the rats were randomly divided into sham operation control group, model control group, evodiamine low(40 mg · kg-1) and high(80 mg· kg-1) dose groups. Evodiamine was intragastric administration for 14 consecutive days, and the sham operation or control rats were given with distilled water. After consecutive 14 d, the neointimal hyperplasia degree was observed by histopathological alterations and by calculating the proliferating cell nuclear antigen(PCNA) positive cells expression percentage in the common carotid arter-y. The levels of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) in plasma of rats were measured respectively. The mRNA expressions of PCNA, endothelial nitric oxide synthase(eNOS) and SM α-actin in carotid artery wall were analyzed separately by real time RT-PCR. RESULTS: The neointimal hyperplasia was very serious, as evidenced by thickened neointima and narrowed lumen in carotid artery balloon-injured rats (model control group). Compared with the model control group, evodiamine 40 and 80 mg · kg-1 administration could significantly ameliorate the histopathology of common carotid artery, decrease the positive expression rate of PCNA, but increase the levels of NO, cGMP in plasma of rats, downregulate the PCNA mRNA expression and upregulate mRNA expressions of eNOS and SM α-actin, respectively. CONCLUSION: Evodiamine can depress the vascular neointimal hyperplasia induced by artery endothelia rubbing in rats, the mechanism may be related, at least partly, to the promotion of NO production.

Effects of Combination Therapy with Celecoxib and Doxycycline on Neointimal Hyperplasia and Inflammatory Biomarkers in Coronary Artery Disease Patients Treated with Bare Metal Stents

PURPOSE: Cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 play a key role in the pathogenesis of in-stent restenosis. We investigated the effect of a short-term therapy of celecoxib, a COX-2 inhibitor, with or without doxycycline, an MMP inhibitor, after coronary stenting on inflammatory biomarkers and neointimal hyperplasia. MATERIALS AND METHODS: A total of 75 patients (86 lesions) treated with bare metal stents were randomized into three groups: 1) combination therapy (200 mg celecoxib and 20 mg doxycycline, both twice daily), 2) celecoxib (200 mg twice daily) only, and 3) non-therapy control. Celecoxib and doxycycline were administered for 3 weeks after coronary stenting. The primary endpoint was neointimal volume obstruction by intravascular ultrasound (IVUS) at 6 months. The secondary endpoints included clinical outcomes, angiographic data, and changes in blood levels of inflammatory biomarkers. RESULTS: Follow-up IVUS revealed no significant difference in the neointimal volume obstruction among the three treatment groups. There was no difference in cardiac deaths, myocardial infarctions, target lesion revascularization or stent thrombosis among the groups. Blood levels of high-sensitivity C-reactive protein, soluble CD40 ligand, and MMP-9 varied widely 48 hours and 3 weeks after coronary stenting, however, they did not show any significant difference among the groups. CONCLUSION: Our study failed to demonstrate any beneficial effects of the short-term therapy with celecoxib and doxycycline or with celecoxib alone in the suppression of inflammatory biomarkers or in the inhibition of neointimal hyperplasia. Large scale randomized trials are necessary to define the role of anti-inflammatory therapy in the inhibition of neointimal hyperplasia.

Comparison of bare metal stent and paclitaxel-eluting stent using a novel rat aorta stent model

The purpose of our study was to create a novel rat aorta stent implantation model. Stainless steel bare metal stents (BMS) or paclitaxel-eluting stents (PES) were implanted in male Sprague-Dawley rats (BW 400 +/- 20 g). Two and four weeks after stent implantation, the aorta were collected, fixed with 2% glutaraldehyde, and cut into two segments. One segment was used for scanning electron microscopy analysis to evaluate re-endothelialization, and the other segment was used to calculate the neointimal area. At 2 weeks after stenting, the appearance of neointimal hyperplasia was less in the PES group than in the BMS group. At 4 weeks after stenting, no significant difference in neointimal hyperplasia was observed between two groups. On the other hand, the PES group showed more thrombus formation and less re-endothelialization compared to the BMS group. This study demonstrated the ability of a novel rat model of aorta stenting via a common carotid artery to measure the efficacy and safety of commercially available drug-eluting stents.

Suppression of Neointimal Hyperplasia by External Application of Cilostazol-Eluting Film at Anastomotic Sites in a Canine Model / 日本心臓血管外科学会雑誌

Neointimal hyperplasia is the principal mechanism of graft failure in coronary artery bypass surgery. Systemic administration of cilostazol has been reported to suppress neointimal hyperplasia in some vascular injury models. We sought to deliver cilostazol locally in an attempt to augment its beneficial effect to inhibit neointimal hyperplasia at an anastomotic site. We examined whether the external application of a novel cilostazol-eluting film can inhibit neointimal hyperplasia in a vascular anastomosis model. Canine femoral artery graft interposition was performed in 20 beagle dogs, assigned to 4 groups of 5 dogs each : a graft interposition without copolymer of L-lactide and ε-caprolactone (P (LA/CL) ) film (control group) and groups with P (LA/CL) film containing cilostazol of either 10 mg, 40 mg, or 80 mg doses. All the cilostazol-eluting film with 10 mg, 40 mg, and 80 mg dose groups had a reduced intima/media ratio compared to the control group (0.15±0.03, 0.11±0.03, and 0.12±0.03, vs. 0.31±0.03, <i>p</i><0.05). Immunohistochemical analyses for proliferating cell nuclear antigens revealed reduced cellular proliferating activity associated with decreased α-actin positive cells in the cilostazol-eluting film groups compared to the control group. External application of cilostazol-eluting film can inhibit neointimal hyperplasia, at least in part, by inhibiting smooth muscle cell proliferation in the intima.

Progress on microRNAs and their roles in vascular diseases / 国际外科学杂志

MicroRNAs as a novel class of endogenous gene regulators at post-transcription level have been found to play important roles in many biological processes including cellular differentiation, proliferation, apoptosis and regulation of development. The biological mechanisms of microRNAs involved in the pathogenesis of various diseases have been revealed gradually. This paper reviews the Current situation and progress on the microRNAs and their roles in vascular diseases.

Alagebrium Chloride, a Novel Advanced Glycation End-Product Cross Linkage Breaker, Inhibits Neointimal Proliferation in a Diabetic Rat Carotid Balloon Injury Model

BACKGROUND AND OBJECTIVES: Vascular perturbation induced by advanced glycation end-products (AGEs) leads to progression of atherosclerosis, plaque instability, and vascular inflammation, which results in a higher risk of neointimal proliferation. Here we investigated the inhibitory effect of alagebrium chloride (ALT-711), a breaker of AGE-based cross links, on neointimal proliferation in a carotid artery balloon injury model in diabetic rats induced by streptozotocin (STZ). MATERIALS AND METHODS: Rat aortic vascular smooth muscle cells (RASMCs) were treated with 1-100 microM of alagebrium added 24 hours before the addition of AGEs. This in vivo study was done using 8-week-old male rats that were injected intraperitoneally with 80 mg/kg STZ. Sixteen weeks later, the diabetic rats were treated with 10 mg/kg alagebrium for 4 weeks, after which carotid artery balloon injury was induced. After 4 weeks, the animals were sacrificed for histological analysis. RESULTS: Proliferation of RASMCs was significantly inhibited in alagebrium-treated cells. Alagebrium dose-dependently inhibited AGE-mediated formation of reactive oxygen species (ROS), extracellular signal-regulated kinase phosphorylation, and cyclooxygenase-2 expression. The cellular mechanisms of AGE-induced connective tissue and extracellular matrix expression were decreased in the alagebrium-treated group. This in vivo study shows that expression of AGE receptors and neointima hyperplasia are significantly suppressed in balloon-injured rats treated with alagebrium. CONCLUSION: Alagebrium treatment in diabetic rats significantly inhibits neointimal hyperplasia after carotid balloon injury due to its inhibition of intracellular ROS synthesis, which results in inhibition of RASMCs proliferation.

Mechanisms of restenosis after vascular reconstruction / 国际外科学杂志

Restenosis is the major causee which leads to arterial occlusion and graft failure after vascular reconstruction.Thus to clarif the mechanism of restenosis is of great importance to prevent and treat postprocedural restenosis and improve long-term graft patency.Current studies on restenosis focus on elastic recoil,thrombosis,inflammatory reaction,neointimal hyperplasia and vascular remodeling,herein,we reviewed literatures.

Effect of Udenafil on Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia in Rat Carotid Artery Injury Model

BACKGROUND AND OBJECTIVES: Neointimal hyperplasia, which was caused by smooth muscle cell proliferation, was noted to occur after performing percutaneous coronary intervention. Phosphodiesterase type 5 (PDE5) inhibitor has been shown to inhibit smooth muscle cell proliferation. Udenafil is one of the PDE5 inhibitors, and it is also expected to inhibit smooth muscle cell proliferation and reduce neointimal hyperplasia. We investigated the effect of udenafil on the smooth muscle cell proliferation and neointimal hyperplasia that occurs after balloon injury in the carotid arteries of rats. MATERIALS AND METHODS: Smooth muscle cells were treated with 1 mM, 100 micrometer, 10 micrometer, 1 micrometer and 100 nM concentrations of udenafil. The viability of the smooth muscle cells was evaluated by MTT assay. The carotid arteries of rats were injured with a balloon catheter. Udenafil (100 micrometer, 10 micrometer and 1 micrometer) was applied on the carotid artery adventitia after balloon injury. At 21 days after treatment, the carotid arteries were harvested and stained with H & E. The neointima and media area were measured with a computerized image analysis program. RESULTS: In the in vitro experiment, treatment with 1 mM udenafil reduced smooth muscle cell viability by 68.8+/-4.42% compared to the control group. In the balloon injured rat carotid artery, treatment with 100 micrometer udenafil reduced the neointima area by 71.8% compared to the control group. CONCLUSION: Udenafil administration effectively inhibited smooth muscle cell proliferation and it reduced neointimal hyperplasia in the balloon-injured rat carotid artery.

The Origin of Neointimal Smooth Muscle Cells in Transplant Arteriosclerosis from Recipient Bone-marrow Cells in Rat Aortic Allograft / 华中科技大学学报（医学）（英德文版）

In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts.Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P＜0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.

The neointimal hyperplasia effect of erythropoietin on carotid artery injury model of rat / 대한내과학회지

BACKGROUN: Neointimal hyperplasia is major cause of instent restenosis in coronary artery and stenosis in arteriovenous fisula for hemodialysis. Erythropoietin is known to show proliferative effect on vascular smooth muscle cells in vitro study, but there is few in vivo study. This study investigated the effect of erythropoietin given subcutaneously to injured carotid arteries of rats on neointimal hyperplasia. METHODS: Sprague-Dawley rat underwent common carotid artery (CCA) balloon injury. Erythropoietin was given subcutaneously to balloon-injured rats (n=6) in 100 U/Kg/week by dividing three times. The control group (n=6) was treated with normal saline only. Two weeks later, the lumen and the neointimal area were obtained and compaired. Actively proliferating cells of neointimal area were observed by microscopy using PCNA staining method. RESULTS: Luminal area was 0.233 +/- 0.073 mm2, 0.112 +/- 0.047 mm2 in the control and the erythropoietin treated group respectively. The area was significantly smaller in the treatment group (p<0.05). Neointimal area and neointimal area/medial area ratio were 0.148 +/- 0.029 mm2, 0.226 +/- 0.056 mm2 and 1.169 +/- 0.146 min, 1.953 +/- 0.465 min in the control and the erythropoietin treated group respectively (p<0.05), but no significant difference was observed in medial area between the control and the erythropoietin treated group. Actively proliferating cells of neointimal area were more commonly observed in erythropoietin treated group. CONCLUSION: Erythropoietin increases neointimal cell proliferation in the rat carotid artery injury model. So, erythropoietin treatment may contributes to the development of arteriovenous fistula stenosis caused by neointimal hyperplasia but, further studies are required to elucidate the mechanism and the effect of erythropoietin at various time and dose in neointimal hyperplasia.

Inhibition of Neointimal Hyperplasia by External Radiation in Rat Carotid Injury Model-The Possible Role of Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1-

BACKGROUND AND OBJECTIVES: Despite significant improvement in the field of angioplasty, restenosis remains a major obstacle to the long-term success of the procedure. Radiation can effectively inhibit neointimal hyperplasia by causing the arrest of mitosis during cell division and limiting proliferation by reducing the number of regenerating clonal progenitors. Balloon injury could induce the cell adhesion molecule, ICAM-1 and VCAM-1, on SMCs and regenerating endothelial cells (ECs). ICAM-1 and/or VCAM-1 may play a role in the progression of neointimal hyperplasia induced by balloon injury and external radiation may effectively inhibit neointimal hyperplasia by attenuating their expression. The purpose of this study was to examine the effect of external radiation against ICAM-1 and VCAM-1 on neointimal hyperplasia after balloon injury in rat carotid arteries. MATERIAL AND METHODS: A standardized carotid balloon catheter arterial injury was produced in 51 rats and external beam radiation with doses from 5-20 Gy were delivered in 28 rats (radiation treated group) at 24 hours after injury. To investigate the effect of the external radiation on neointimal hyperplasia, the intima area and the intima/medial area of arteries were measured at day 14 after injury. The expressions of ICAM-1 and VCAM-1 at day 2, day 7, and day 10 after injury were studied in control group and radiation treated group by immunohistochemistry. RESULTS: Means of intimal area and intima/medial ratio in radiation treated group were significantly lower than those in control group and significantly reduced with increasing radiation dosage. At day 2 after injury, medial SMCs of injury group extensively expressed ICAM-1, while it was focally expressed with 10 Gy radiation treated group. At day 7 and day 10 after injury, ICAM-1 expression on medial SMCs was attenuated and neointimal ICAM-1 expression was increased. As compared with control group, ICAM-1 expression after radiation was weak and focal just around the internal elastic lamina. At 2 days after injury, medial SMCs moderately expressed VCAM-1, which was weakly and focally expressed with 10 Gy radiation treated group. At day 7 and day 10 after injury, focal expression of VCAM-1 was noted around the internal elastic lamina, but there was no VCAM-1 expression on neointima with radiation. CONCLUSION: External radiation after carotid arterial injury may potentially inhibit SMC proliferation and neointimal hyperplasia, and balloon injury-induced or upregulated expressions of ICAM-1 and VCAM-1 may be attenuated with external radiation.

Effect of Local Administration of Lovastatin on Preventing Neointimal Hyperplasia in the Rat Carotid Artery Injury Model

BACKGROUND AND OBJECTIVES: ovastatin, a HMG-CoA reductase inhibitor, is known to show antiproliferative effects on VSMC after vessel injury, but a large amount of the drug is needed orally for this purpose. This study investigated the effects of lovastatin given locally to injured carotid arteries of rats on reducing neointimal hyperplasia. MATERIALS AND METHOD: Lovastatin was given perivascularly to balloon-injured carotid arteries of 21 rats in 1 microM to the low-dose group, and 30 microM to the high-dose group. The control group was treated with pluronic gel only. Two weeks later, the lumen area, neointimal areas and the number of actively proliferating cells were obtained and compared. RESULTS: eointimal area was 0.113+/-0.032 mm2, 0.065+/-0.017 mm2, 0.072+/-0.017 mm2 in the control, low-dose and high-dose groups respectively. The area was significantly smaller in the treatment groups (p<0.05), but no significant difference was observed between the treatment groups. The number of actively proliferating cells per mm2 of neointimal area were 714.5+/-227.4, 688.4+/-333.7, and 1526.3+/-744.0 in the groups respectively, and the number was significantly high in the high-dose group (p<0.05). CONCLUSION: Local administration of lovastatin is effective in reducing neointimal hyperplasia after vascular injury, but extremely high doses are not needed locally for this purpose.

Effect of Local Administration of Lovastatin on Preventing Neointimal Hyperplasia in the Rat Carotid Artery Injury Model

BACKGROUND AND OBJECTIVES: ovastatin, a HMG-CoA reductase inhibitor, is known to show antiproliferative effects on VSMC after vessel injury, but a large amount of the drug is needed orally for this purpose. This study investigated the effects of lovastatin given locally to injured carotid arteries of rats on reducing neointimal hyperplasia. MATERIALS AND METHOD: Lovastatin was given perivascularly to balloon-injured carotid arteries of 21 rats in 1 microM to the low-dose group, and 30 microM to the high-dose group. The control group was treated with pluronic gel only. Two weeks later, the lumen area, neointimal areas and the number of actively proliferating cells were obtained and compared. RESULTS: eointimal area was 0.113+/-0.032 mm2, 0.065+/-0.017 mm2, 0.072+/-0.017 mm2 in the control, low-dose and high-dose groups respectively. The area was significantly smaller in the treatment groups (p<0.05), but no significant difference was observed between the treatment groups. The number of actively proliferating cells per mm2 of neointimal area were 714.5+/-227.4, 688.4+/-333.7, and 1526.3+/-744.0 in the groups respectively, and the number was significantly high in the high-dose group (p<0.05). CONCLUSION: Local administration of lovastatin is effective in reducing neointimal hyperplasia after vascular injury, but extremely high doses are not needed locally for this purpose.