Inhibition of NF-kappa B pathway leads to deregulation of epithelial-mesenchymal transition and neural invasion in pancreatic cancer.

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial-mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell-cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion.

Covering the proximal nerve stump with chondroitin sulfate proteoglycans prevents traumatic painful neuroma formation by blocking axon regeneration after neurotomy in Sprague Dawley rats.

OBJECTIVE: Neuropathic pain caused by traumatic neuromas is an extremely intractable clinical problem. Disorderly scar tissue accumulation and irregular and immature axon regeneration around the injury site mainly contribute to traumatic painful neuroma formation. Therefore, successfully preventing traumatic painful neuroma formation requires the effective inhibition of irregular axon regeneration and disorderly accumulation of scar tissue. Considering that chondroitin sulfate proteoglycans (CSPGs) can act on the growth cone and effectively inhibit axon regeneration, the authors designed and manufactured a CSPG-gelatin blocker to regulate the CSPGs' spatial distribution artificially and applied it in a rat model after sciatic nerve neurectomy to evaluate its effects in preventing traumatic painful neuroma formation. METHODS: Sixty female Sprague Dawley rats were randomly divided into three groups (positive group: no covering; blank group: covering with gelatin blocker; and CSPG group: covering with the CSPG-gelatin blocker). Pain-related factors were evaluated 2 and 8 weeks postoperatively (n = 30). Neuroma growth, autotomy behavior, and histological features of the neuromas were assessed 8 weeks postoperatively (n = 30). RESULTS: Eight weeks postoperatively, typical bulb-shaped neuromas did not form in the CSPG group, and autotomy behavior was obviously better in the CSPG group (p < 0.01) than in the other two groups. Also, in the CSPG group the regenerated axons showed a lower density and more regular and improved myelination (p < 0.01). Additionally, the distribution and density of collagenous fibers and the expression of α-smooth muscle actin were significantly lower in the CSPG group than in the positive group (p < 0.01). Regarding pain-related factors, c-fos, substance P, interleukin (IL)-17, and IL-1ß levels were significantly lower in the CSPG group than those in the positive and blank groups 2 weeks postoperatively (p < 0.05), while substance P and IL-17 remained lower in the CSPG group 8 weeks postoperatively (p < 0.05). CONCLUSIONS: The authors found that CSPGs loaded in a gelatin blocker can prevent traumatic neuroma formation and effectively relieve pain symptoms after sciatic nerve neurotomy by blocking irregular axon regeneration and disorderly collagenous fiber accumulation in the proximal nerve stump. These results indicate that covering the proximal nerve stump with CSPGs may be a new and promising strategy to prevent traumatic painful neuroma formation in the clinical setting.

A free vein graft cap influences neuroma formation after nerve transection.

INTRODUCTION: : Neuroma formation is a major problem in nerve surgery and consensus about its prevention has not been reached. It has been suggested that vein covering can reduce neuroma formation in transected nerves. In this article, the Authors propose an easy and novel method of covering by nerve stump capping with a free vein graft. METHODS: : Neuroma-like lesions were created on the rat thigh sectioning the femoral nerve above its division in 16 animals. The proximal nerve stump was invaginated into the lumen of a 1.5 cm long femoral free vein graft on the right side, and the vein was closed on itself by microsurgical sutures to form a cap for the nerve stump. On the left side acting as the control neuroma, the nerve was cut and left uncovered. Histological and immunohistochemical assessment was used to quantify the degree of neuroma formation. RESULTS: : Significant differences were found in both neuroma size and axon-glia organization between the treated and control sides indicating that free vein graft capping reduced neuroma formation in comparison to uncovered nerve stumps. CONCLUSIONS: : Our results confirm that vein-covering of a transected nerve stump can be effective in reducing neuroma formation. Moreover, unlike previous works that buried the nerve into an adjacent vein left in place, our experiments showed that also the use of a free vein graft cap can hinder neuroma formation. Although translation of rat experiments to the clinics should be dealt with caution, our data suggest a careful clinical use of the technique. (c) 2009 Wiley-Liss, Inc. Microsurgery, 2009.

Delayed tumor onset in transgenic mice fed a low-folate diet.

BACKGROUND: Transgenic mice carrying the human T-lymphotropic virus type 1 tax1 (transactivator) gene develop peripheral nerve sheath tumors with well-characterized times of onset and tissue involvement. PURPOSE AND METHODS: To evaluate the effect of dietary folic acid on age at tumor onset and on the concentration of folate in tissues and tumors, we bred heterozygous transgenic mice and systematically assigned their offspring at weaning (within litters) to a 2 x 2 x 2 factorial arrangement. The three variables studied were 1) the tax1 gene (presence or absence), 2) gender (male or female), and 3) dietary level of folic acid (0.11 or 11.34 mumol folic acid per kilogram of controlled amino acid-based diet). Blood and tissues were collected from tumor-bearing transgenic mice (prior to cachexia) and from nontransgenic littermates, matched whenever possible for gender and diet. RESULTS: Transgenic mice fed a diet containing 0.11 mumol of folic acid per kilogram developed tumors significantly later (92.8 +/- 6.4 days) than did those fed a diet containing 11.34 mumol of folic acid per kilogram (71.9 +/- 3.9 days). Folate concentrations in tumors of mice fed the low-folate diet were approximately one third those in tumors of mice fed the higher folate diet. Brain folate concentrations in mice fed the low-folate diet were less than one half those in mice fed the higher folate diet. CONCLUSION: Results show that the onset of spontaneous tumors can be delayed by feeding mice the lowest level of folate adequate to meet nutritional requirements for normal growth. IMPLICATION: Transgenic animal models of human disease offer great potential for evaluating the role of micronutrients in human carcinogenesis.

Reduction of N-nitroso-N-ethylurea-induced neurogenic tumors by X-radiation: a life-span study in rats.

Whole-body X-irradiation after neonatal injection with N-nitroso-N-ethylurea (ENU) significantly reduced the incidence of induced neurogenic tumors in inbred HMT rats kept for their complete life-span. After administration of 10 mg ENU/kg and 1.25 Gy X-radiation, the incidence of schwannomas but not of gliomas was reduced as compared to the incidence in rats given 10 mg ENU/kg only. In contrast, after administration of 4 mg ENU/kg, 1.25 Gy reduced the incidence of gliomas but not of schwannomas. Administration of 1.25 Gy alone induced a remarkably high incidence of rats with neurogenic tumors (20%). Latency of tumor detection was not significantly affected by radiation. Among the most frequently occurring nonneurogenic tumors, squamous cell carcinomas were reduced in incidence by treatment with ENU, 1.25 Gy X-radiation, or both combined. No treatment affected the incidence of pituitary or mammary tumors. There was a preponderance of ovarian tumors in rats given 4 mg ENU/kg + 1.25 Gy. An incidental finding was the occurrence of granular cell tumors in 7 rats from different treatment groups.

Treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (ENU)-induced neoplasms of the nervous system in Sprague-Dawley rats.

The ability of field bean protease inhibitor (FBPI) to inhibit ethylnitrosourea (ENU)-induced tumours of the nervous system of Sprague-Dawley rats was investigated. Groups of 1-day-old rats were injected intraperitoneally (i.p.) with neurocarcinogenic amounts of ENU and a few hours later, one group was treated i.p. with 80 mg of FBPI per kg body weight. This treatment was carried out three times a week for the first month and five times a week for the next month. Animals were killed when they were neurologically ill and their neural tissues were assessed for lesions. Those FBPI-treated rats which showed no illness were also killed to terminate the experiment about 8 weeks after the last rat of the control group was affected with paralysis. The neural tumours induced in all groups were predominantly large tumours found in the cerebrum of the rats. ENU-treated rats showed a 100% incidence of nervous system tumours with a mean time of manifestation of neurological symptoms of 282 days, which was significantly shorter in comparison to that noted in the FBPI-treated group. The latter group showed an incidence of 58.3%, i.e. a significant reduction of 41% in the incidence of neural tumours, as well as a lower mean value for the number of tumours per rat. All these aspects indicated that FBPI is a potential neurooncopreventive agent. A neural tumour incidence of 100% in the rats treated with heat-inactivated FBPI confirmed that the tumour suppressive activity of FBPI is related to its protease inhibitory activity.

Neuroma formation and prevention by fascicle ligation in the rat.

Light and electron microscopic studies of the cut sciatic nerve in the rat showed typical neuroma formation. If the nerve was cut and the proximal segment was ligated as a whole, the neuromatous process appeared to be even more severe than that in the simply cut nerve. When the perineurium of the cut nerve was kept intact by fascicle ligation, the axonal regenerative process seemed to be altered significantly, and less neuroma formation occurred. The mechanism by which fascicle ligation limits axonal regeneration is unknown, but could involve peripheral as well as central mechanisms. Such a limitation of neuroma formation by fascicle ligation as was found in the rat might also apply to painful human neuromas.

Centrocentral anastomosis in the prevention and treatment of painful terminal neuroma. An experimental study in the rat.

In this experimental study, microsurgical centrocentral anastomosis was applied to an experimental model of painful terminal neuroma resulting from left sciatic nerve section in the rat. The anastomosis consisted of end-to-end suturing of the sciatic nerve fascicles to the tibial branch, with the interposition of a nerve graft taken from the same anastomosed fascicle. As a control parameter for the experiment, the autotomy which follows sciatic nerve section in the rat was evaluated. Autotomy is considered an objective indication of abnormal sensations that are provoked by the formation of a terminal neuroma. Histological study of the proximal stump of the sciatic nerve was also performed. The observation period was 10 weeks. The study demonstrates that centrocentral anastomosis reduces the size of the neuroma formation and the incidence of autotomy.

Ineffectiveness of dietary folic acid supplementation on the incidence of lipomyelomeningocele: pathogenetic implications.

OBJECT: Periconceptual folic acid supplementation is effective in myelomeningocele prevention. The relationship between folic acid and lipomyelomeningocele (LMM) and the overall incidence of this occult form of spina bifida has never been studied. The objectives of this study were to determine the impact of dietary folic acid supplementation on the incidence of LMM and to measure its overall incidence. METHODS: In a retrospective population-based study the authors calculated the incidence of LMM in Nova Scotia between 1985 and 2001. Because of changes in public policy during this period, there are three intervals defined in relation to the treatment of the food supply with folic acid: 1) prior to folic acid fortification (1985-1994); 2) postsupplementation but prefortification (1995-1998); and 3) postfortification. The overall incidence of LMM in Nova Scotia between 1985 and 2001 was 16 per 100,000 live births or one case per 6121 live births. Its incidence between 1985 and 1994 was 15 per 100,000 live births, and between 1995 and 1998 it was 12 per 100.000 live births (relative risk [RR] 0.82, 95% confidence interval [CI] 0.31-2.22; p = 0.7). Between 1999 and 2001, the incidence of LMM was 29 per 100,000 live births, which was not significantly different from that between 1995 and 1998 (RR 2.41. 95% CI 0.79-7.36; p = 0.11) or between 1985 and 1994 (RR 1.98, 95% CI 0.86-4.56; p = 0.1). CONCLUSIONS: The overall incidence of LMM between 1985 and 2001 in Nova Scotia was 16 per 100,000 live births and has not been reduced by dietary folic acid supplementation. This finding provides epidemiological evidence that the embryogenesis of LMM is fundamentally different from that of myelomeningocele.

Comparative study of neuroma formation in the rat sciatic nerve after CO2 laser and scalpel neurectomy in combination with milliwatt CO2 laser "sealing".

Nerve transections of rat sciatic nerves were performed to compare scalpel neurectomy to CO2 laser neurectomy. Additionally, the effect of milliwatt CO2 laser application to the proximal nerve ending was studied. Animals were sacrificed at 1 to 9 weeks, and the neuromas were examined histologically and measured. Scalpel transections produced typical neuromas. Laser transections produced neuromas characterized by multinucleated giant cells and carbonaceous debris. There was no difference in the size of the neuromas produced by either method of transection. Milliwatt CO2 laser application to the proximal stump did not affect the size or histology of resultant neuroma formation regardless of the method of transection.

Implantation of peripheral neural stump into muscle and its effect on the development of posttraumatic neuroma.

Dissection of the peripheral nerve is associated inherently with the development of posttraumatic neuroma on the end of the proximal stump of damaged nerve. It is unfavourable, as it causes pain on the side of dissected nerve and additionally it is an obstacle in case of secondary surgical reconstruction of the nerve. Among many methods of treating stumps of dissected nerves in order to avoid the development of neuromas, it is recommended to sew the proximal stump of dissected nerve into the venter of adjacent skeletal muscle. The present study is an evaluation of histological changes occurring at the borderline between nerve and muscle after implanting the proximal stump of dissected femoral nerve into the venter of skeletal muscle. The experiments were carried out in 30 WAG rats in which sciatic nerves were cut and the proximal end of the nerve was introduced into the venter of the adjacent muscle. The nerves with muscular fragments were obtained for histological analysis at 3, 5, 10, 20 and 40 days. In no specimen signs of developing posttraumatic neuroma were found.

[Effect of PS-K on experimental neurogenic tumors in rats induced by ethylnitrosourea].

The effect of PS-K as immunomodulator was studied in Fischer rats bearing neurogenic tumors induced transplacentally by ENU. 114 rats were divided into control and PS-K treated groups. all rats were subjected to an autopsy at death and all of central nervous system were examined macroscopically. Final neurogenic tumor incidence in PS-K treated groups was significantly lower than that in the control group, but distribution of these neurogenic tumors and number of neurogenic tumors in a rat remained unchanged between the control and the each PS-K treated groups. Survival time of PS-K treated groups did not increase than the control group. From these experiments, it might be that induction of neurogenic tumors may be suppressed by administration of PS-K.

Preventing neurovascular invasion in desmoid tumors.

Desmoid tumors or aggressive fibromatoses are rare, non-encapsulated, infiltrative and locally aggressive tumors originating from deep musculo-aponeurotic structures. Traditionally, preferred treatment method for desmoid tumors is wide local excision. Depending on the side and type of resection, the reported local recurrence rates range from 15 to 77%. Similarly, in our institution there is a significant recurrence rate (24%) in patients who underwent surgery for desmoid tumor. After several recurrences, amputation may be inevitable following repeating vascular and nerve reconstructions. There is a need for a nonviable barrier in order to prevent the invasion of the viable tumor to the neurovascular structures which are also viable tissues. Depending on this need, we present two cases that we used synthetic vascular graft in their operations to cover neurovascular structures in order to prevent tumor invasion. For patients who are not suitable for radiotherapy and the neurovascular structures need to be secured because of the risk of local recurrence, this method can prevent possible future invasion of vessels and nerves.

The role of different methods of nerve ablation in prevention of neuroma.

BACKGROUND: The aim of this study was to compare the incidence of neuroma formation and neuropathic pain following different techniques of nerve ablation in a rat sural nerve model. METHODS: Rat sural nerve was subjected to four different techniques of ablation with standardized creation of a 1-cm gap (n = 15 in each group). These included nerve avulsion, transection and burying in muscle, transection and folding of nerve, and transection alone. Animals were killed after 3 months. Explanted nerves were sectioned and stained with Masson trichrome and S-100 stain against neural tissue. The maximal neural cross-sectional area and neural-to-connective tissue ratio was quantified. Quantitative reverse-transcriptase polymerase chain reaction (n = 5) was used to analyze relative mRNA expression of ciliary neurotrophic factor and calcitonin gene-related peptide. RESULTS: Neural cross-sectional area was statistically increased (p < 0.05) compared with controls in folded, muscle buried, and transected specimens but decreased in avulsed specimens. The neural-to-connective tissue ratio was statistically decreased in the avulsed group. Relative mRNA expression of ciliary neurotrophic factor was lowest in muscle buried (4 percent of control) (p < 0.05) and avulsed specimens (15 percent of control) (p < 0.05) and higher in folded (52 percent of control) and transected specimens (75 percent of control). Relative mRNA expression of calcitonin gene-related peptide was highest in folded specimens (302 percent of control) (p < 0.05). CONCLUSIONS: Folding and transection lead to increased histologic evidence of neuroma formation, whereas folding leads to neuropathic pain, assayed by calcitonin gene-related peptide expression. Avulsion and muscle burying are preferable techniques for nerve ablation and inhibit nerve regeneration, evidenced by decreased ciliary neurotrophic factor expression. Avulsion offers an alternative to muscle burying when there is no muscle in the vicinity to bury the transected nerve.

Neuroma formation in a rat median nerve model: influence of distal stump and muscular coating.

BACKGROUND: The purpose of this study was to investigate neuroma formation in a rat median nerve model. METHODS: In three groups, the median nerve was exposed and a gap was created. In the first group, a short gap of 1 cm (n = 12) was created; in the second, a long gap of 2 cm (n = 12) was created in the nerve. Another group was used to analyze the development of neuroma formation when the proximal stump was buried in adjacent muscle with an additional gap of 2 cm (n = 12). The use of different lengths should allow one to gain information about dilution effects of distal stump factors that may contribute to neuroma formation. Nine months later, specimens were gathered and histologically analyzed. The cross-sectional areas of neuromas were measured and the neural/connective tissue ratios were estimated. RESULTS: The cross-sectional areas demonstrated that neuroma formation was significantly higher in the short-gap group than in the long-gap group, and smallest in the muscle-covered group. The percentage of neural tissue was highest in the muscle-covered and long-gap groups and lowest in the short-gap group. CONCLUSIONS: These results demonstrate an association between neuroma formation and distal stump distance. This observation may be explained by the factors originating from the distal stump that were blocked when the proximal nerve stump was completely buried in the muscle. For clinical application, the authors recommend not only burying the proximal stump in a muscle but also surgically augmenting the gap between the proximal and distal stumps.

Perineural invasion of cutaneous malignancies.

BACKGROUND: Perineural invasion is an important mode of tumor spread and is associated with increased aggressiveness and a propensity for recurrence among cutaneous malignancies. OBJECTIVE: To review the pathogenesis, diagnosis, and treatment of cutaneous tumors exhibiting perineural invasion. METHODS: This article is based on a review of the medical literature concerning tumors with perineural involvement. RESULTS: This article describes the clinical signs and histologic features of cutaneous malignancies exhibiting perineural involvement. CONCLUSION: Appropriate patient care mandates consideration of perineural invasion in the evaluation of cutaneous tumors. As the majority of patients present without symptoms of neural involvement, physicians must be vigilant in the search for this type of tumor spread.

Influence of nerve stump transplantation into a vein on neuroma formation.

The objective of this animal study was to investigate the influence of nerve stump transposition into a vein on neuroma formation. In 24 rats the femoral nerve was severed and the proximal nerve stump was transposed into the lumen of the femoral vein on one side. On the other side, the nerve was severed and left in place. The distal nerve stump was shortened to knee level on both sides. In group 1, the bloodstream was released; in group 2, the segment of the femoral vein containing the nerve stump was excluded from circulation. Histological assessment was performed 8 months later. There were significant differences between the treatment and control sides with respect to neuroma size, endoneural architecture, neural-tissue-to-connective-tissue ratio, and myelination of axons. These data suggest that nerve transposition into a vein could inhibit the formation of classic neuroma.

Prevention and treatment of painful neuromas of the superficial radial nerve by the end-to-side nerve repair concept: an experimental study and preliminary clinical experience.

This article studies the utilization of the end-to-side neurorrhaphy concept in the prevention and treatment of painful neuromas. A total of 20 rats were divided into 2 groups (10 rats per group). In group A, the tibial nerve was divided and left lying in the subcutaneous tissue. In group B, the cut ends of the tibial nerve were sutured to the adjacent peroneal nerve in an end-to-side fashion. Evaluation was performed 90 days after nerve injury. For group A, the proximal end of the tibial nerve formed a "classic" neuroma and the distal end showed a degenerated nerve. In group B, the proximal end of the tibial nerve formed a "non-classic" neuroma and the nerve healed into the peroneal nerve with continuity of the epineurium of the 2 nerves. The distal end of the tibial nerve in group B showed evidence of axonal regeneration. Preliminary clinical experience utilizing the same technique in the prevention and treatment of painful neuromas of the superficial radial nerve is presented and other techniques of nerve-to-nerve implantation are discussed.

Study of nerve regeneration in centrocentral anastomosis.

Nerve regeneration was studied in a model of centrocentral anastomosis (CCA) performed on the sciatic nerve of the rat. Experimental CCA was made by suturing the proximal end of the peroneal branch on the proximal end of the sural branch, placing between them a peroneal nerve graft (Group I, 20 rats) or a silicone chamber (Group II, 12 rats). Nerve grafts had a length of 5 mm and silicone chambers 7 mm. In six silicone chambers an 1 mm nerve graft was placed in the centre of the tube. In group I animals anterograde degeneration was studied by cutting the graft 60 days after surgery. In group II, nerve regeneration was studied 2, 4 and 8 weeks after surgery. Results indicate that in CCA: 1) regenerated axons coming from one nerve end grow into the graft but do not cross the contralateral suture line; 2) regeneration is poorer in silicone chambers than in nerve grafts; and 3) in silicone chambers regeneration is related to time. The reduction in the regenerative capability in CCA seems to be related to the alteration of nerve sprouts aiming for the peripheral targets.