Ectodysplasin A in Biological Fluids and Diagnosis of Ectodermal Dysplasia.

The tumor necrosis factor (TNF) family ligand ectodysplasin A (EDA) is produced as 2 full-length splice variants, EDA1 and EDA2, that bind to EDA receptor (EDAR) and X-linked EDA receptor (XEDAR/EDA2R), respectively. Inactivating mutations in Eda or Edar cause hypohidrotic ectodermal dysplasia (HED), a condition characterized by malformations of the teeth, hair and glands, with milder deficiencies affecting only the teeth. EDA acts early during the development of ectodermal appendages-as early as the embryonic placode stage-and plays a role in adult appendage function. In this study, the authors measured EDA in serum, saliva and dried blood spots. The authors detected 3- to 4-fold higher levels of circulating EDA in cord blood than in adult sera. A receptor binding-competent form of EDA1 was the main form of EDA but a minor fraction of EDA2 was also found in fetal bovine serum. Sera of EDA-deficient patients contained either background EDA levels or low levels of EDA that could not bind to recombinant EDAR. The serum of a patient with a V262F missense mutation in Eda, which caused a milder form of X-linked HED (XLHED), contained low levels of EDA capable of binding to EDAR. In 2 mildly affected carriers, intermediate levels of EDA were detected, whereas a severely affected carrier had no active EDA in the serum. Small amounts of EDA were also detectable in normal adult saliva. Finally, EDA could be measured in spots of wild-type adult or cord blood dried onto filter paper at levels significantly higher than that measured in EDA-deficient blood. Measurement of EDA levels combined with receptor-binding assays might be of relevance to aid in the diagnosis of total or partial EDA deficiencies.

Histologic evaluation of the effects of Emdogain gel on injured root apex in rats.

INTRODUCTION: This study investigated the effects of Emdogain gel (EMD) on the injured open apex within periapical lesions. METHODS: Periapical lesions were induced in rats by opening the pulp chambers of the mandibular first molars and filing the apical foramen through the distal root canal with #25 K-files to make an open apex. The teeth were exposed to the oral environment for 7 days. Then we irrigated the distal root canals and divided them into EMD-treated and propylene glycol alginate-treated groups. The rats were killed 7, 14, and 28 days after treatment and examined histochemically. RESULTS: In the EMD-treated rats, more cells expressed transforming growth factor-ß1 or bone morphogenetic protein-2 at 7 days after treatment, and the regeneration of cementum and bone was observed around the root apex at 14 days after treatment. Conversely, in the propylene glycol alginate-treated group, few cells expressed transforming growth factor-ß1 or bone morphogenetic protein-2, and apical periodontal tissue recovery was rarely seen within the periapical lesions throughout the experiment. CONCLUSIONS: These results suggest that EMD does not irritate injured periapical tissue and may create a favorable environment that promotes the healing of destroyed periapical tissues.

Evaluation of tumor necrosis factor α blockade on early tendon-to-bone healing in a rat rotator cuff repair model.

PURPOSE: The purpose was to determine whether systemic tumor necrosis factor α (TNF-α) blockade can improve rotator cuff healing in a rat model. METHODS: One hundred twenty Lewis rats underwent unilateral detachment and repair of the supraspinatus. Rats were randomized into 2 groups. The experimental group received injections of pegylated soluble tumor necrosis factor receptor type I (3.0 mg/kg every other day for 3 doses). The control group received saline solution on the same dosing schedule. At 2, 4, and 8 weeks, 20 animals in each group were killed (4 for histologic assessment and 16 for biomechanical testing). Outcomes included qualitative histologic assessment to determine new fibrocartilage formation and collagen fiber organization. Immunohistochemical staining was performed to localize TNF-α, ED1 and ED2 macrophages, and tartrate-resistant acidic phosphatase. Biomechanical testing was performed to determine the ultimate load to failure, stiffness, cross-sectional area, and ultimate stress to failure. RESULTS: Qualitative assessments of histology showed that the experimental group had more cartilage formation at 4 weeks but not at 2 or 8 weeks. There was less TNF-α staining in the experimental group at 4 and 8 weeks, and there were fewer ED1 macrophages at 4 weeks compared with controls. The ultimate load to failure was greater in the experimental group compared with controls at 2 weeks (13.3 ± 2.6 N v 11.2 ± 2.7 N, P = .05) and at 4 weeks (21.7 ± 4.6 N v 18.5 ± 2.1 N, P = .04). The experimental group also had a higher stiffness at 2 weeks (7.2 ± 2.3 N/mm v 5.8 ± 1.4 N/mm, P = .04) and at 4 weeks (10.5 ± 2.7 N/mm v 8.4 ± 1.7 N/mm, P = .01). There were no differences in any biomechanical variable at 8 weeks. CONCLUSIONS: TNF-α blockade can improve the biomechanical strength of tendon-bone healing in a rat rotator cuff model at early time points, which corresponded with modest qualitative improvements in histology. However, these differences were not maintained at 8 weeks. CLINICAL RELEVANCE: TNF-α blockade may influence rotator cuff tendon healing.

Reaction of endogenous progenitor cells in a rat model of posttraumatic syringomyelia.

OBJECT: Endogenous stem cells theoretically could replace lost tissue and repair deficits caused by syringes. In this study the authors quantitatively examined 1) whether neural progenitor cells exist in an adult rat model of posttraumatic syringomyelia (PTS); 2) and if so, how long an active population of progenitor cells can persist; 3) whether the cell population's location is associated with the syrinx; 4) the degree of differentiation of the progenitor cells; and 5) the phenotypic fate of the progenitor cells. METHODS: Wistar rats were divided into intact, sham-operated, and experimental syrinx groups. Animals in each group were equally subdivided according to 4 time points: 7, 14, 28, and 56 days post-syrinx induction. Rats in the experimental syrinx group underwent a C-7 and T-1 laminectomy and then received 0.5 µl of a 24-mg/ml quisqualic acid spinal cord injection at the C-8 level to mimic an excitotoxic injury with an initial cyst, and 10 µl of a 250-mg/ml kaolin injection into the subarachnoid space at the C-8 level to create arachnoiditis. The proliferation, distribution, and differentiation of endogenous progenitor cells were identified immunocytochemically. RESULTS: The authors observed a 20-fold increase in progenitor cells excluding inflammatory cells in the 1st 2 weeks post-syrinx induction. The cells persisted for at least 56 days, and 80% of them were located in the gray matter along the border of cysts. They included neural multipotential progenitor cells, oligodendroglial progenitor cells, and astrocytes. CONCLUSIONS: Data in this study provide evidence for proliferation, distribution, and differentiation of endogenous progenitor cells in a model of PTS in adult rats. These progenitor cells proliferate rapidly, extend for long periods, and are mainly located in the gray matter along the border of syringes. Neural multipotential progenitor cells are expected to be associated with reparative and regenerative mechanisms of PTS. Glial cells are involved in the formation of a glial scar barrier that surrounds the syrinx and may prevent cyst enlargement. The authors' findings suggest that neural progenitor cells play a protective role in PTS.

All-trans retinoic acid results in irregular repair of septa and fails to inhibit proinflammatory macrophages.

All-trans retinoic acid (ATRA) is controversially discussed in emphysema therapy. We re-evaluated ATRA in the elastase model and hypothesised that beneficial effects should be reflected by increased alveolar surface area, elastin expression and downregulation of inflammatory mediators and matrix metalloproteinases (MMPs). Emphysema was induced by porcine pancreatic elastase versus saline in Sprague-Dawley rats. On days 26-37, rats received daily intraperitoneal injections with ATRA (500 µg · kg(-1) body weight) versus olive oil. Lungs were removed at day 38. Rat alveolar epithelial L2 cells were incubated with/without elastase followed by ATRA- or vehicle-treatment, respectively. ATRA only partially ameliorated structural defects. Alveolar walls exhibited irregular architecture: increased arithmetic mean thickness, reduction in surface coverage by alveolar epithelial cells type II. ATRA only partially restored reduced soluble elastin. It tended to increase the ratio of ED1(+):ED2(+) macrophages. Bronchoalveolar lavage (BAL) cells exhibited a proinflammatory state and high expression of interleukin-1ß, cytokine-induced neutrophil chemoattractant-1, tumour necrosis factor-α, nuclear factor-κB, MMP-2, MMP-9, MMP-12, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in emphysema, with ATRA exerting only few effects. MMP-7 was highly induced by ATRA in healthy but not in emphysematous lungs. ATRA reduced both MMP-2 and TIMP-1 activity in BAL fluid of emphysematous lungs. ATRA-therapy may bear the risk of unwanted side-effects on alveolar septal architecture in emphysematous lungs.

Persistent behavioral impairments and neuroinflammation following global ischemia in the rat.

Cognitive deficits associated with cardiac arrest have been well documented; however, the corresponding deficits in animal models of global ischemia have not been comprehensively assessed, particularly after long-term, clinically relevant survival times. We exposed male Sprague-Dawley rats to 10 min of bilateral carotid artery occlusion + systemic hypotension (40-45 mmHg) or sham surgery, and used histopathological assessments for short-term survival animals (16 days) and both behavioral and histopathological assessments for long-term survival animals (270 days). Analyses revealed significant long-term deficits in ischemic animals' learning, memory (T-maze, radial arm maze), working memory (radial arm maze), and reference memory (Morris water maze, radial arm maze) abilities that were not associated with a general cognitive decline. Histological results showed significant increases in glial fibrillary acidic protein, neuron glia 2, OX-42 and ED-1 staining, as well as significant decreases in microtubule-associated protein 2 staining and cornu ammonis area 1 (CA1) cell counts 16 days post-ischemia. The pattern at 270 days was similar, but notably there was a persistent elevation of ED-1 staining, suggesting recent cell death as well as significant atrophy of CA1. Whereas previous work has primarily reported transient changes in behavior after global ischemia, this study describes disturbances in several different functional domains following CA1 cell loss at clinically relevant survival times. Moreover, the histopathological outcome is suggestive of a spontaneous repopulation of CA1, but this was not sufficient to offset the behavioral impairments arising from the ischemic insult.

Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone(r)) osteogenesis: a study on biopsies from human jaws.

OBJECTIVES: Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone(s)), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. MATERIAL AND METHODS: Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodelling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). RESULTS: Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone(s) granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone(s) and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2-immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. CONCLUSIONS: We postulate that NanoBone(s) has osteoconductive and biomimetic properties and is integrated into the host's physiological bone turnover at a very early stage.

Effect of betamethasone on the degree of macrophage recruitment and nerve growth factor receptor p75 immunoreaction during recovery of the sciatic nerve after injury: an experimental study in rats.

PURPOSE: This study was designed to explain our previous findings of beneficial effects of betamethasone given perioperatively on decreasing the incidence of neurosensory disturbance after sagittal split osteotomy and improving functional recovery after crush injury to rat sciatic nerves. We analysed the pattern of macrophage recruitment and expression of nerve growth factor p75. MATERIAL AND METHODS: The sciatic nerve was crushed in each of 42 animals by tying the nerve against a glass rod for 30s. Half the rats were given betamethasone and half were not. The effect of betamethasone was evaluated immunohistochemically in a double blind manner after 2, 7 and 17 days using antibodies against macrophage marker (ED1) and p75. RESULTS: We found an initial and significant decrease in the number of macrophages recruited after two days in the group treated with betamethasone compared with controls (p=0.001). By 7 days there were significantly more macrophages in the steroid group than in the control group (p=0.001). There was however, a tendency for the number of p75R to be higher in the in the steroid group but the difference was not significant. At 17 days, there were significantly fewer macrophages in the steroid group (p=0.008) than in the control. CONCLUSION: We conclude that the beneficial effect of a moderate perioperative dose of betamethasone on recovery of a nerve is reflected in the recruitment of macrophages but to only a small extent in expression of p75.

ORMOSIL nanoparticles as a non-viral gene delivery vector for modeling polyglutamine induced brain pathology.

Studies have shown the presence of expanded polyQ containing proteins in brain cells related to Huntington disease (HD) and other poly-glutamine disorders. We report the use of organically modified silica (ORMOSIL) nanoparticles as an efficient non-viral gene carrier in an effort to model brain pathology associated with those disorders induced by expanded polyQ peptides. In experiment 1, plasmids expressing Hemaglutinin-tagged polypeptides with 20 glutamine repeats (Q20) or with extended 127-glutamine repeats (Q127) were complexed with ORMOSIL nanoparticles and injected twice (2 weeks apart) into the lateral ventricle of the mouse brain. Fourteen days post-injection of Q127, immunocytochemistry revealed the presence of the characteristic nuclear and cytoplasmic Q127 aggregates in numerous striatal, septal and neocortical neuronal cells as well as ubiquitin-containing aggregates indicative of the neuronal pathology. The mice receiving Q127 showed a marked increase in the reactive GFAP (+) astrocytes in striatum, septum and brain cortex, further indicating the neurodegenerative changes, accompanied by motor impairments. In experiment 2, plasmids Q20 or Q127 were complexed with ORMOSIL and were injected into the brain lateral ventricle or directly into the striatum of adult rats. In both routes of transfection, Q127 induced the appearance of reactive GFAP (+) astrocytes and activated ED1 antigen expressing microglia. An increase in the size of the lateral ventricle was also observed in rats receiving Q127. In transgenic mouse polyQ models, extensive pathologies occur outside the nervous system and the observed brain pathologies could reflect developmental effects of the toxic polyQ proteins. Our experiments show that the nervous tissue restricted expression of poly Q-extended peptides in adult brain is sufficient to evoke neuropathologies associated with HD and other polyQ disorders. Thus, nanotechnology can be employed to model pathological and behavioral aspects of genetic brain diseases in mice as well as in other species, providing a novel research tool for in vivo testing of single or multi-gene therapies.

Histone deacetylase inhibitors exhibit anti-inflammatory and neuroprotective effects in a rat permanent ischemic model of stroke: multiple mechanisms of action.

The pathophysiology of cerebral ischemia involves multiple mechanisms including neuroinflammation mediated by activated microglia and infiltrating macrophages/monocytes. The present study employed a rat permanent middle cerebral artery occlusion (pMCAO) model to study effects of histone deacetylase (HDAC) inhibition on ischemia-induced brain infarction, neuroinflammation, gene expression, and neurological deficits. We found that post-pMCAO injections with HDAC inhibitors, valproic acid (VPA), sodium butyrate (SB), or trichostatin A (TSA), decreased brain infarct volume. Postinsult treatment with VPA or SB also suppressed microglial activation, reduced the number of microglia, and inhibited other inflammatory markers in the ischemic brain. The reduction in levels of acetylated histone H3 in the ischemic brain was prevented by treatment with VPA, SB, or TSA. Moreover, injections with HDAC inhibitors superinduced heat-shock protein 70 and blocked pMCAO-induced down-regulation of phospho-Akt, as well as ischemia-elicited up-regulation of p53, inducible nitric oxide synthase, and cyclooxygenase-2. The motor, sensory, and reflex performance of pMCAO rats was improved by VPA, SB, or TSA treatment. The beneficial effects of SB and VPA in reducing brain infarct volume and neurological deficits occurred when either drug was administrated at least 3 h after ischemic onset, and the behavioral improvement was long-lasting. Together, our results demonstrate robust neuroprotective effects of HDAC inhibitors against cerebral ischemia-induced brain injury. The neuroprotection probably involves multiple mechanisms including suppression of ischemia-induced cerebral inflammation. Given that there is no effective treatment for stroke, HDAC inhibitors, such as VPA, SB, and TSA, should be evaluated for their potential use for clinical trials in stroke patients.

A heterogeneous immunofluorescence staining for laminin-1 and related basal lamina molecules in the dorsal root ganglia following constriction nerve injury.

The bodies of primary sensory neurons and their satellite glial cells (SGCs) are limited by the basal laminae from extracellular matrix of the dorsal root ganglia (DRG). The basal laminae displayed uniform immunofluorescence staining for laminin-1 in the sections of rat intact (naive) DRG. A proximal or distal ligature of the spinal nerves resulted in a heterogeneous immunostaining for laminin-1 around neuron-SGC units in the sections of the corresponding DRG. The pattern of irregular laminin-1 immunofluorescence was more extensive in the ipsilateral than the contralateral DRG of the operated rats. The immunofluorescence for laminin-1 exactly coincided with binding of Concanavalin-A as well as immunostaining for type IV collagen in both naive DRG and DRG affected by nerve ligature. Nidogen immunostaining decreased or fully disappeared at the surface of the SGCs consistently with immunofluorescence staining for laminin-1, but retained or increased in the endothelial cells and ED-1 positive cells invaded the DRG affected by nerve ligature. The results indicate an alteration of the content of basal laminae surrounding the bodies of primary sensory neurons and their SGSs following nerve constriction injury. A modulation of the basal laminae may be related with other cellular and molecular alterations related with peripheral neuropathic pain, for example, expansion of sympathetic sprouts.