Exposure Duration Is a Determinant of the Effect of Sinusoidal Electromagnetic Fields on Peak Bone Mass of Young Rats.

We proposed a three-step strategy to obtain the optimal therapeutic parameters, which is composed of large-scale screening at cellular level, verification in animal experiments, and confirmation by a clinical trial. The objective of the current study was to test the feasibility of our strategy. Newborn rat calvarial osteoblasts were treated by 50 Hz 1.8 mT sinusoidal electromagnetic fields (SEMFs) with 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h/days, respectively. The osteogenic differentiation and maturation of the osteoblast were assayed and compared to obtain the optimal duration. One-month-old growing rats were then treated by the same SEMFs with 0.5, 1.5, and 2.5 h/days, respectively, and the peak bone mass was analyzed after 2 months. It was found that the optimal exposure duration to promote the osteogenic differentiation and maturation of osteoblasts was 1.5 h/days, judging by the increasing degrees of ALP activity, calcified nodules formed, the gene and protein expression levels of Runx-2, BMP-2, and Col-I, as well as the expression levels of signaling proteins of the BMP-2/Smad1/5/8 pathway. The highest increase of peak bone mass after 2 months was also obtained by 1.5 h/days, judging by the results of X-ray dual-energy absorptiometry, mechanical property analysis, micro-CT scanning, and serum bone turnover marker examinations. The above results indicated that exposure duration is a determinant for the therapeutic effect of EMFs, and the optimal therapeutic effects only can be obtained by the optimal exposure duration.

Primary cilium is required for the stimulating effect of icaritin on osteogenic differentiation and mineralization of osteoblasts in vitro.

OBJECTIVE: Icaritin, one effective metabolite of Herba Epimedii-derived flavonoid icariin, has a strong osteogenic activity. However, its action mechanism remains unclear. Since primary cilia have been shown to play a pivotal role in regulating the osteogenesis, we hypothesized primary cilia are indispensable in mediating icaritin osteogenic effect. MATERIALS AND METHODS: Primary rat calvarial osteoblasts were transfected with siRNA1 targeting intraflagellar transport protein 88 (IFT88), a protein required for ciliogenesis, to prevent formation of primary cilium and were treated with 10-6 M icaritin. RESULTS: Alkaline phosphatase (ALP) activity was significantly increased after 3 days in cells transfected with scrambled siRNA control and treated by icaritin (SC+I group) compared to cells transfected with scrambled siRNA control only (SC group). ALP activity after IFT88 siRNA1 transfection and icaritin treatment (siRNA1+I group) was significantly lower than that of SC+I group. Formation of ALP positively stained colonies after 6 days, osteocalcin secretion after 9 days and formation of calcified nodules after 12 days displayed a similar tendency among the three groups. mRNA expression of osteogenesis-related genes ALP, BMP-2, COL1α, RUNX-2 and OSX after 24 h was significantly increased in SC+I group, but was not different with SC group in siRNA1+I group. Protein levels of BMP-2, COL1α, RUNX-2 and OSX after 48 h showed the similar tendency with gene expression. CONCLUSION: Primary cilia are important in mediating icaritin-stimulated osteogenic differentiation and may be a novel target for pharmacological therapies for bone loss.

Icariin attenuates hypoxia-induced oxidative stress and apoptosis in osteoblasts and preserves their osteogenic differentiation potential in vitro.

OBJECTIVES: Icariin, a prenylated flavonol glycoside isolated from traditional Chinese medicinal herb of the genus Epimedium, has been demonstrated to be a potential alternative therapy for osteoporosis, and its action mechanism so far has been mainly attributed to its phytoestrogenic property. As blood supply to bone is considerably reduced with ageing and by the menopause, we hypothesized that icariin treatment would reduce bone loss by preventing ischaemia-induced hypoxic damages to bone. MATERIALS AND METHODS: To investigate effects of icariin treatment on cultured rat calvarial osteoblasts exposed to hypoxic conditions (2% oxygen). RESULTS: Compared to normoxic control, cell viability decreased with time to 50% by 48 h in the hypoxic group, and icariin attenuated the reduction, dose dependently, with 10(-6) and 10(-5)  m concentrations showing significant protective effects. Icariin also inhibited increase of lactate dehydrogenase activity in culture media. Measurements on oxidative stress, cell cycling and cell survival indicated that icariin protected osteoblasts by reducing production of reactive oxygen species and malondialdehyde, increasing superoxide dismutase activity, arresting the cell cycle and inhibiting apoptosis. Icariin also preserved osteogenic differentiation potential of the hypoxic cells in a dose-dependent manner, compared to the hypoxia alone group, as revealed by increased levels of RUNX-2, OSX and BMP-2 gene expression, alkaline phosphatase activity, and formation of mineralized nodules. CONCLUSIONS: Our results demonstrated that icariin attenuated oxidative stress and apoptosis and preserved viability and osteogenic potential of osteoblasts exposed to hypoxia in vitro, and suggested that its anti-osteoporotic effect may be attributed to its anti-hypoxic activity and phytoestrogenic properties.

Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area.

Injury to the growth plate is common and yet the injured cartilage is often repaired with undesirable bony tissue, leading to bone growth defects in children. Using a rat tibial growth plate injury model, our previous studies have shown sequential inflammatory, fibrogenic, osteogenic and bone maturation responses involved in the bony repair. However, it remains unclear whether there is progressive accumulation of bone within the injury site and any potential degenerative changes at the adjacent non-injured area of the growth plate. This study examined effects of growth plate injury on the structure, composition and some cellular and molecular changes at the injury site and adjacent uninjured area. Micro-CT analysis revealed that while the bone volume within the injury site at day 14 was small, the bone bridge was considerably larger at the injury site by 60 days post-injury. Interestingly, formation of bone bridges in the adjacent uninjured area was detected in 60% of injured animals at day 60. Immunohistochemical analyses revealed reduced chondrocyte proliferation (PCNA labelling) but increased apoptosis (nick translation labelling) in the adjacent uninjured area. RT-PCR analysis on adjacent uninjured growth plate tissue found increased expression of osteocalcin at day 60, differential expression of apoptosis-regulatory genes and alterations in genes associated with chondrocyte proliferation/differentiation, including Sox9 and IGF-I. Therefore, this study has demonstrated progressive changes in the structure/composition of the injury site and adjacent uninjured area and identified cellular and molecular alterations or degeneration in adjacent uninjured growth plate in response to injury.

Pathobiology and prevention of cancer chemotherapy-induced bone growth arrest, bone loss, and osteonecrosis.

Cancer chemotherapy has been recognized as one severe risk factor that influences bone growth and bone mass accumulation during childhood and adolescence. This article reviews on the importance of this clinical issue, current understanding of the underlying mechanisms for the skeletal defects and potential preventative strategies. Both clinical and basic studies that appeared from 1990 to 2010 were reviewed for bone defects (growth arrest, bone loss, osteonecrosis, and/or fractures) caused by paediatric cancer chemotherapy. As chemotherapy has become more intensive and achieved greater success in treating paediatric malignancies, skeletal complications such as bone growth arrest, low bone mass, osteonecrosis, and fractures during and/or after chemotherapy have become a problem for some cancer patients and survivors particularly those that have received high dose glucocorticoids and methotrexate. While chemotherapy-induced skeletal defects are likely multi-factorial, recent studies suggest that different chemotherapeutic agents can directly impair the activity of the growth plate and metaphysis (the two major components of the bone growth unit) through different mechanisms, and can alter bone modeling/remodeling processes via their actions on bone formation cells (osteoblasts), bone resorption cells (osteoclasts) and bone "maintenance" cells (osteocytes). Intensive use of multi-agent chemotherapy can cause growth arrest, low bone mass, fractures, and/or osteonecrosis in some paediatric patients. While there are currently no specific strategies for protecting bone growth during childhood cancer chemotherapy, regular BMD monitoring and exercise are have been recommended, and possible adjuvant treatments could include calcium/vitamin D, antioxidants, bisphosphonates, resveratrol, and/or folinic acid.

Damage and recovery of the bone marrow microenvironment induced by cancer chemotherapy - potential regulatory role of chemokine CXCL12/receptor CXCR4 signalling.

The bone marrow microenvironment houses haematopoietic stem cells (HSC), mesenchymal stem cells (MSC) and their progeny, supports haematopoiesis, osteogenesis, osteoclastogenesis, and adipogenesis. It plays a key role in maintaining homeostatic production of erythroid, myeloid or lymphoid cells, appropriate bone mass and bone health throughout life. Through cell-cell adhesion and chemotactic axes, a reciprocal inter-dependent relationship exists between these two cell lineages. Following chemotherapy-induced myelosuppression observed in cancer patients, HSCs are induced to enter into the cell cycle in order to re-establish the damaged microenvironment. These cells not only have the capacity to mobilize to the peripheral blood, but the ability to repopulate the marrow cavity as required. However, depending on the dosage and length of chemotherapy treatment, complications in bone and bone marrow recovery occur. This may manifest as marrow haematopoietic depletion, high marrow fat content, reduced bone formation and aggravated osteoclastic bone resorption. Although the molecular and cellular mechanisms governing injured states of the marrow microenvironment are yet to be fully elucidated, many reports have demonstrated the CXCL12/CXCR4 axis plays an important role in regulating the two cell lineages. Their interaction maintains bone marrow homeostasis and orchestrates its regeneration following chemotherapy. This review explores movement of MSC and HSC, haematopoiesis, commitment of osteoblasts, osteoclasts, and adipocytes, as well as the major signalling pathways that regulate these cellular processes under chemotherapy-treated conditions. This review also discusses molecular targets that are being used clinically or are currently under investigation for preserving the bone marrow microenvironment during or enhancing recovery after chemotherapy.

Roles of COX-2 and iNOS in the bony repair of the injured growth plate cartilage.

Growth plate injuries often lead to bone growth defects, which primarily occur due to bony repair at injury sites. Bony repair is preceded by an injury-induced inflammatory response, which could play a role in regulating the repair process. Here, roles of two inflammatory mediators, cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), in the injury responses were analysed by examining their gene expression and effects of blocking their activities, respectively, with celecoxib and aminoguanidine during 2 days prior to and until 7 days after injury in a rat tibial growth plate injury model. Quantitative RT-PCR assays revealed upregulated expression of COX-2 on days 1 and 4 and iNOS on day 1. Histological analysis of injury sites revealed significant reductions in inflammatory infiltrate (particularly neutrophils) on day 1 in treated groups compared to saline control. While bony tissue proportions at injury sites were unaffected by either treatment, mesenchymal tissue proportions were larger but cartilaginous tissue proportions were smaller on day 8 (though statistically insignificant), and bone remodelling appeared delayed with a smaller bone marrow proportion on day 14 in both treatment groups. These findings suggest that COX-2 and iNOS mediate injury-induced inflammatory response, and may play a role in enhancing mesenchymal cell differentiation to cartilaginous cells and in promoting bone remodelling during bony repair of growth plate injury sites. Furthermore, increased expression of cartilage-related (collagen-2, collagen-10, SOX-9) and bone-related molecules (osteocalcin, cbfalpha-1) suggest involvement of both endochondral and direct bone formation mechanisms during bony repair.

Differential effects of endogenous brain-derived neurotrophic factor on the survival of axotomized sensory neurons in dorsal root ganglia: a possible role for the p75 neurotrophin receptor.

After peripheral nerve injury, axotomized sensory neurons in dorsal root ganglia (DRG) undergo apoptosis and up-regulate brain-derived neurotrophic factor (BDNF). We tested whether endogenous BDNF plays any role in the survival of axotomized sensory neurons using in vitro and in vivo models. In the in vitro model, treatment with BDNF antibody significantly reduced apoptosis of sensory neurons in DRG explants from both adult and neonate rats and adult mice cultured for 48 h. Consistently, exogenous BDNF increased the percentage of apoptotic neurons in the DRGs from mice. The effects of the BDNF antibody and BDNF were not seen in DRGs from p75NTR(-/-) mice. In the in vivo model, sciatic nerve transection in neonatal rats decreased the total number of neurons in the injured DRG and treatment with antiserum to BDNF significantly exaggerated the loss of DRG neurons. Numbers of sensory neurons expressing BDNF and p75NTR in cultured DRGs increased but that expressing TrkB decreased. In contrast, sciatic nerve transection in vivo reduced the numbers of neurons expressing both p75NTR and TrkB but increased the numbers of cells expressing BDNF, 1 and 7 days after the surgery. These results suggest that BDNF may have differential effects on the survival of sensory neurons depending on the expression of p75NTR. While endogenous BDNF induced apoptosis of axotomized sensory neurons through p75NTR in vitro where more neurons expressed p75NTR, it prevented apoptosis in vivo where fewer neurons expressed p75NTR after sciatic nerve transection.

Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats.

With the intensified use of chemotherapy and improved survival rates for childhood malignancies, it has become increasingly apparent that some children or adult survivors show poor bone growth and develop osteoporosis. As a step to investigate underlying mechanisms, this project examined short-term effects in rats of chemotherapy agent 5-fluorouracil (5-FU) on cell proliferation, apoptosis, and bone formation at tibial growth plate cartilage and its adjacent bone-forming region metaphysis. In addition, since insulin-like growth factor (IGF-I) is important for bone growth, we examined whether IGF-I pretreatment would potentially protect growth plate cartilage and bone cells from chemotherapy damage. Two days after a single high dose of 5-FU injection, proliferation of growth plate chondrocytes and metaphyseal osteoblasts/preosteoblasts was dramatically suppressed, and apoptosis was induced among osteoblasts and preosteoblasts. As a result, there was a reduction in the chondrocyte number and zonal height at the proliferative zone and a decline in the number of osteoblasts and preosteoblasts on the metaphyseal trabecular bone surface. At day 2, no obvious deleterious effects were observed on the height of the growth plate hypertrophic zone and the bone volume fraction of the metaphyseal primary spongiosa trabeculae. At day 10, while cell proliferation and growth plate structure returned to normal, there were slight decreases in trabecular bone volume, body length increase, and tibial length. While pretreatment with 1-week IGF-I systemic infusion did not attenuate the suppressive effect of 5-FU on proliferation in both growth plate and metaphysis, it significantly diminished apoptotic induction in metaphysis. These results indicate that growth plate cartilage chondrocytes and metaphyseal bone cells are sensitive to chemotherapy drug 5-FU and that IGF-I pretreatment has some anti-apoptotic protective effects on metaphyseal bone osteoblasts and preosteoblasts.

Roles of growth factors in chemotherapy-induced intestinal mucosal damage repair.

Chemotherapy agents induce apoptotic cell death and loss of cell proliferation in the intestinal crypt epithelium, resulting in intestinal mucosal damage called "mucositis". Small intestinal mucositis is characterized structurally by crypt loss and villus atrophy, and functionally by absorptive and barrier impairments. The increased use of chemotherapy in cancer treatment and the clinical importance of the intestinal mucositis as a common side effect have stimulated more active research into understanding the pathophysiology of intestinal mucositis and developing agents for preventing or treating this condition. Rodent studies have shown that, following the chemotherapy-induced initial apoptosis and loss of crypt cell proliferation, many different growth factors or their receptors are upregulated locally at the crypts, preceding or coinciding with the epithelial hyperproliferative repair response. Aiming to reduce crypt cell apoptotic sensitivity to cytotoxic chemotherapy and/or to enhance crypt epithelial proliferative repair, several exogenous growth factor treatments have been tested, either preclinically and/or clinically, and are showing promise for their efficacy or safety in preventing or treating chemotherapy-induced mucositis. These tested growth factors include keratinocyte growth factor, interleukin-11, transforming growth factor beta, milk-derived growth factor extract, macrophage/granulocyte colony stimulating factors, and glucagon-like peptide 2. Further research on the basic and discovery levels and subsequent translational studies are needed to understand more about chemotherapy-induced intestinal mucositis and to identify candidates of growth factors or other agents that will potentially prevent or treat chemotherapy-induced mucositis more effectively, specifically, safely, and practically in chemotherapy patients.

Lack of effects of transforming growth factor-alpha gene knockout on peripheral nerve regeneration may result from compensatory mechanisms.

Transforming growth factor-alpha (TGF-alpha), previously identified as a major member of the epidermal growth factor (EGF) family of growth factors, plays a role in proliferation, differentiation, and survival of neuronal and glial precursors and is implicated in development of the nervous system. However, its roles in nerve injury-induced responses remain obscure. The current study examined roles of endogenous TGF-alpha in peripheral nerve regeneration using sciatic nerve injury models with TGF-alpha knockout mice. Three weeks after a sciatic nerve crush, no significant differences were found between TGF-alpha wild-type and mutant mice in the number of retrogradely labeled L5 dorsal root ganglion (DRG) sensory neurons and L5 spinal cord motor neurons and in the morphology of myelinated regenerating nerve fibers, indicating that TGF-alpha is not essential for sensory and motor nerve regeneration. To assess a possible functional redundancy among TGF-alpha-related ligands in response to a nerve injury, mRNA expression of the EGF family was analyzed by RT-PCR in L4/L5 DRG pools and distal degenerating sciatic nerve segments after sciatic nerve ligation. Prior to and 1 day after ligation, there was a higher level of EGF-R mRNA in DRGs and in nerve in TGF-alpha null mice compared to wild types, and there was an induction of ligand amphiregulin mRNA in DRGs in mutant mice in place of the TGF-alpha upregulation present in wild types. These results indicate that TGF-alpha gene knockout does not affect peripheral nerve regeneration, probably due to a functional redundancy within the EGF family through a compensatory expression mechanism at both the receptor and ligand levels in TGF-alpha knockout mice.

Growth retardation, duodenal lesions, and aberrant ileum architecture in triple null mice lacking EGF, amphiregulin, and TGF-alpha.

BACKGROUND AND AIMS: Mice lacking epidermal growth factor (EGF), transforming growth factor alpha, and amphiregulin were used to identify roles for these EGF receptor (EGF-R) ligands in gastrointestinal development and mucosal integrity. METHODS: Gastrointestinal tract development was examined in knockout mice and correlated with expression of EGF-R protein and EGF family members throughout the gut. Crossfostering experiments addressed roles of maternal- and neonatal-derived ligands in pup growth and intestinal development. Cysteamine-induced ulceration in EGF(-/-) mice was used to examine its role in mucosal cytoprotection. RESULTS: Neonatal mice lacking all 3 ligands were growth retarded, even when reared by wild-type dams; conversely, lack of maternal ligands transiently impaired wild-type pup growth. Triple null neonates displayed spontaneous duodenal lesions, and ileal villi were truncated and fragile with reduced cellular proliferation in the crypts. However, maturation of digestive enzymes was unaffected. Adult EGF(-/-) mice displayed more severe lesions in response to cysteamine treatment compared with wild-type counterparts, although triple null mice were not more susceptible to dextran sulfate sodium-induced colitis, suggesting a differential role for these ligands in the injury response. CONCLUSIONS: EGF-R ligands are required for development and mucosal maintenance in mouse small intestine. Both maternal and neonatal sources of growth factors are required for optimal pup growth.

Expression of B7 costimulatory molecules by cells infiltrating the colon in experimental colitis induced by oral dextran sulfate sodium in the mouse.

BACKGROUND AND AIM: T-cell activation, mediated by the interaction with major histocompatibility complex (MHC)-peptide complexes and B7 costimulatory molecules on antigen-presenting cells, is an essential event in the pathogenesis of inflammatory bowel disease (IBD). We investigated the expression of B7 costimulatory molecules on cells in the colon in an experimental mouse model of IBD to determine whether the B7/ligand interaction could provide a target for therapeutic intervention in IBD. METHODS: Experimental colitis was induced in mice by oral consumption of water substituted with 5% dextran sulfate sodium (DSS). Mice (n=4) were killed 1, 2, 3, 4 and 7 days after commencing DSS consumption, and colonic tissue was collected and examined immunohistochemically for T cells, B cells, macrophages and cells expressing B7-1 or B7-2. RESULTS: Compared to control mice drinking water, macrophage numbers in the colonic epithelium were elevated sevenfold by day 1 and T cells were elevated threefold by day 3 following commencement of DSS consumption. Numbers of infiltrating B7-positive (B7+) cells were not significantly elevated until day 7 when B7-1+, B7-2+ cells and macrophages were increased 20-fold compared to normal mice. CONCLUSION: These results demonstrate that an initial and rapid infiltration of the colonic epithelium by B7-negative macrophages is followed by an infiltration of T cells and subsequent upregulation of the B7 costimulatory molecules potentiating the inflammatory reaction in this disease model. These results suggest an intervention strategy based on the blockade of the B7-costimulatory axis could find application in the treatment of inflammatory bowel disease.

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents.

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.

Predisposition to colonic dysplasia is unaffected by continuous administration of insulin-like growth factor-I for twenty weeks in a rat model of chronic inflammatory bowel disease.

BACKGROUND: Insulin-like growth factor-I (IGF-I) is currently under evaluation for the treatment of a variety of chronic disease conditions. We investigated the safety of long-term IGF-I administration in a rat model of inflammatory bowel disease which predisposes to the development of dysplasia. METHODS: Chronic consumption of dextran sulphate sodium (DSS) by rats manifests a colitis with dysplastic features. Rats consumed 2% DSS for 4 weeks when pumps were implanted to deliver either vehicle or IGF-I for 15 or 20 weeks while rats continued to consume DSS. Features of colitis and dysplasia were assessed at kill. RESULTS: Compared to vehicle, 20 weeks IGF-I significantly increased body weight by 19% and total gut weight by 43%. Colonic crypt depth, proliferative compartment, labelling index, dysplasia, neoplasia and other indices of colitis were not significantly affected. CONCLUSIONS: Twenty weeks administration of IGF-I to rats induced growth of the intestine but did not affect the severity of experimentally-induced colitis or the incidence or progression of colonic dysplasia.

Increased expression of HGF and c-met in rat small intestine during recovery from methotrexate-induced mucositis.

Chemotherapy or radiotherapy often cause mucosal damage in the gut (gut mucositis) in cancer patients. As a step to investigate mechanisms underlying subsequent intestinal repair, we have examined the expression profiles of hepatocyte growth factor (HGF) and its receptor c-met, two molecules previously implicated in tissue repair, in comparison to the histopathological and proliferative changes in a rat model of methotrexate-induced small intestinal mucositis. Histological analysis of the intestinal specimens revealed crypt loss and villus atrophy with damage maximal on day 5 after methotrexate injection, and normalization of mucosal structure commencing on day 6. Crypt cell proliferation was decreased dramatically on day 3, normalized on day 4 and up-regulated on days 5 and 6. HGF and c-met protein/mRNA expression was up-regulated between days 4 and 7, with the mRNA co-localizing to the crypt and lower villus epithelium. Therefore, following methotrexate injection, a decrease in crypt cell proliferation preceded histological damage, and conversely, crypt cell hyperproliferation preceded mucosal regeneration. Up-regulation of HGF and c-met coincided with crypt hyperproliferation and mucosal recovery, suggesting a role for HGF in intestinal repair following acute injury. The crypt epithelial localization of HGF and c-met implies an autocrine or paracrine mechanism of HGF action.

Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats.

Injury to peripheral nerves often results in chronic pain which is difficult to relieve. The mechanism underlying the pain syndrome remains largely unknown. In previous studies we showed that neurotrophins are up-regulated in satellite cells around sensory neurons following sciatic nerve lesion. In the present study, we have examined whether the neurotrophins in the dorsal root ganglia play any role in allodynia after nerve injury. Antibodies to different neurotrophins, directly delivered to injured dorsal root ganglia, significantly reduced (with different time sequences) the percentage of foot withdrawal responses evoked by von Frey hairs. The antibodies to nerve growth factor acted during the early phase but antibodies to neurotrophin-3 and brain-derived neurotrophic factor were effective during the later phase. Exogenous nerve growth factor or brain-derived neurotrophic factor, but not neurotrophin-3, directly delivered to intact dorsal root ganglia, trigger a persistent mechanical allodynia. Our results showed that neurotrophins within the dorsal root ganglia after peripheral nerve lesion are involved in the generation of allodynia at different stages. These studies provide the first evidence that ganglia-derived neurotrophins are a source of nociceptive stimuli for neuropathic pain after peripheral nerve injury.

Cloning of rat betacellulin and characterization of its expression in the gastrointestinal tract.

Betacellulin (BTC) is relatively a more recently discovered member of the EGF family of growth factors. As a prelude to its expression and functional studies in rat models of gut damage/repair, we have cloned rat BTC and examined its expression in the gastrointestinal tract. Rat BTC was found to be nearly identical to mouse betacellulin. A single 3 kb mRNA species was detected by Northern blotting, and ribonuclease protection analysis showed that its expression was ubiquitous but low in abundance throughout the gut. BTC mRNA and protein were found expressed in the gastric surface and upper pit epithelium as well as in some cells of gastric glands. In the jejunum, BTC mRNA and protein were localised to the crypt epithelium and in villous goblet cells. In the colon, BTC mRNA and protein were found produced in crypt and surface epithelium as well as in goblet cells. Taken together, the wide spread expression in the gut epithelium and in mucous cells in particular suggests an important and unique role for BTC in the gastrointestinal tract.

Temporal changes in TFF3 expression and jejunal morphology during methotrexate-induced damage and repair.

Trefoil factor TFF3 has been implicated in intestinal protection and repair. This study investigated the spatiotemporal relationship between TFF3 expression and morphological changes during intestinal damage and repair in a rat model of methotrexate-induced small intestinal mucositis. Intestinal tissues from rats with mucositis were collected daily for 10 days. Mucosal damage was characterized by an initial decrease in cell proliferation resulting in crypt loss, villus atrophy, and depletion of goblet cells, followed by hyperproliferation that lead to crypt and villus regeneration and mucous cell repopulation. TFF3 mRNA levels increased marginally during histological damage, and the cell population expressing TFF3 mRNA expanded from the usual goblet cells to include some nongoblet epithelial cells before goblet cell repopulation. TFF3 peptide, however, was depleted during histological damage and normalized during repair, mirroring the disappearance and repopulation of goblet cells. Although there is no temporal relationship between TFF3 levels and crypt hyperproliferation, confirming the nonmitogenic nature of TFF3, the coincidental normalization of TFF3 peptide with repopulation of goblet cells and mucin production after proliferative overshoot suggests that TFF3 may play a role in the remodeling phase of repair.