Search

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration. SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets., (Copyright © 2022 the authors.)

Lymphovascular invasion (LVI) and perineural invasion (PNI) are 2 important pathologic parameters and need to be accurately assessed in multiple malignancies. Integrin β4, a member of the integrin family, has been reported to be positively expressed in vascular endothelia, peripheral nerves, and a collection of epithelia. However, little is known about the effectiveness of β4 immunostaining on the recognition of LVI and PNI. Herein, we explored the applicability of β4 immunostaining in stomach, thyroid, and breast cancers. Parallel immunostaining of D2-40, CD34, and S-100 was performed as controls for lymphatic endothelia, vascular endothelia, and neural fibers, respectively. The results demonstrated that β4 concurrently stained the lymphatic and vascular endothelia, and the peripheral nerves. Both LVI and PNI were clearly and accurately outlined by β4 immunostaining. β4 was also expressed in the majority of tumor cells, enabling recognition of LVI and PNI encroached by small tumor clusters. In contrast to D2-40 and CD34, β4 staining was not observed in stromal cells, and therefore it facilitated differentiation between the shrinkage cleft and LVI. According to our results, β4 staining strikingly increased the diagnostic accuracy and interobserver concordance for LVI and PNI compared with hematoxylin and eosin staining alone. Finally, the applicability of β4 was confirmed in 9 other types of malignancies, including cancers of the colon, prostate, esophagus, lung, kidney, uterus, tongue, bladder, and liver. Collectively, β4 is a reliable marker for synchronous detection and diagnosis of LVI and PNI.

Olfaction is critical for survival in neonatal mammals. However, little is known about the neural substrate for this ability as few studies of synaptic development in several olfactory processing regions have been reported. Odor information detected in the nasal cavity is first processed by the olfactory bulb and then sent via the lateral olfactory tract to a series of olfactory cortical areas. The first of these, the anterior olfactory nucleus pars principalis (AONpP), is a simple, two layered cortex with an outer plexiform and inner cell zone (Layers 1 and 2, respectively). Five sets of studies examined age-related changes in the AONpP. First, immunocytochemistry for glutamatergic (VGlut1 and VGlut2) and GABAergic (VGAT) synapses demonstrated that overall synaptic patterns remained uniform with age. The second set quantified synaptic development with electron microscopy and found different developmental patterns between Layers 1 and 2. As many of the interhemispheric connections in the olfactory system arise from AONpP, the third set examined the development of crossed projections using anterograde tracers and electron microscopy to explore the maturation of this pathway. A fourth study examined ontogenetic changes in immunostaining for the proteoglycans aggrecan and brevican, markers of mesh-like extracellular structures known as perineuronal nets whose maturation is associated with the end of early critical periods of synaptogenesis. A final study found no age-related changes in the density of vasculature in the peduncle from P5 to P30. This work is among the first to examine early postnatal changes in this initial cortical region of the olfactory system., (© 2019 Wiley Periodicals, Inc.)

Iron (Fe) deficiency (FeD) and manganese (Mn) overexposure (MnOE) may result in several neurological alterations in the nervous system. Iron deficiency produces unique neurological deficits due to its elemental role in central nervous system (CNS) development and myelination, which might persist after normalization of Fe in the diet. Conversely, MnOE is associated with diverse neurocognitive deficits. Despite these well-known neurotoxic effects on the CNS, the influence of FeD and MnOE on the peripheral nervous system (PNS) remains poorly understood. The aim of the present investigation was to examine the effects of developmental FeD and MnOE or their combination on the sciatic nerve of young and adult rats. The parameters measured included divalent metal transporter 1 (DMT1), transferrin receptor (TfR), myelin basic protein (MBP) and peripheral myelin protein 22 (PMP22) expression, as well as Fe levels in the nerve. Our results showed that FeD produced a significant reduction in MBP and PMP22 content at P29, which persisted at P60 after Fe-sufficient diet replenishment regardless of Mn exposure levels. At P60 MnOE significantly increased sciatic nerve Fe content and DMT1 expression. However, the combination of FeD and MnOE produced no marked motor skill impairment. Evidence indicates that FeD appears to hinder developmental peripheral myelination, while MnOE may directly alter Fe homeostasis. Further studies are required to elucidate the interplay between these pathological conditions.

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.

The suprachoroidal region of the eye comprises vascular channels, melanocytes, and thin fibroblasts with elongated cytoplasm that are positioned directly adjacent to the densely collagenous sclera. Morphological similarities between these suprachoroidal fibroblasts and arachnoid cells and perineurial cells have been recognized, but whether these fibroblasts have a perineurial cell-like immunophenotype is not known. To further examine the relationship of these three cell types, we investigated the comparative expression of epithelial membrane antigen (EMA), the tight junction protein claudin-1, glucose transporter-1 (Glut-1), and CD34 in suprachoroidal fibroblasts, arachnoid of the optic nerve sheath, and perineurium of ciliary nerves in eight human eye specimens. Granular, diffuse, and cytoplasmic EMA expression was seen in suprachoroidal fibroblasts, but this was not contiguous with the similar pattern of EMA expression in adjacent perineurium and arachnoid. CD34 expression in suprachoroidal fibroblasts was also seen, similar to arachnoid and perineurium. Claudin-1 and Glut-1 were not consistently expressed in suprachoroidal fibroblasts, distinguishing them from perineurial cells in particular and suggesting that these fibroblasts do not arise directly from adjacent arachnoid or perineurium. Nonetheless, the overlapping morphology and protein expression suggest phenotypic similarities in these cells that protect and support adjacent retina, optic nerve, and peripheral nerve.

Cognitive function declines with age. Such function depends on γ-oscillation in the frontal cortex. Pyramidal neurons, and the parvalbumin-expressing interneurons (PV neurons) that control them, are important for the generation of γ-oscillation. The mechanism by which cognitive function declines is unclear. Perineuronal nets (PNNs) mainly surround the soma and proximal dendrites and axon segments of PV neurons in the cerebral cortex. Previous evidence indicates that PNNs inhibit neural plasticity. If this is true, an increase in the number of neurons surrounded by PNNs or in the thickness or density of the PNNs around neurons could decrease plasticity in the cortex. To determine if an aging-related change in cortical PNNs occurs, we examined the influence of aging on PV neurons and whether Wisteria floribunda agglutinin-positive PNNs differ depending on the cortical area. The results showed that the number of PV neurons/mm 2 did not change in many areas of the cortex as mice aged. In contrast, the number of neurons in the sensory cortex surrounded by PNNs increased as mice aged. Thus, with age, PNN density increases in some cortical areas but not in others. In addition, the expression level of PV protein in PV neurons decreased with aging in the whole cortex. We suggest that decreased expression of PV protein impairs fast spiking in PV neurons. We propose that PNNs surround more neurons as age increases. This aging-related increase in PNNs decreases plasticity in the cerebral cortex and reduces cognitive function. The first step in investigating this proposal would be to determine if PNN density increases with age., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Epithelial sheath neuroma is a rarely recognized but established entity in the medical literature. First described in 2000 by Requena et al, there have only been 7 published cases to date, mostly in female patients and presenting as symptomatic solitary lesions on the back without a known history of trauma. In 2006, Beer et al described and reviewed a dozen cases in which epithelial sheath neuroma-like features were seen in the advent of a surgical procedure, which was termed "re-excision perineural invasion" and attributed to possible eccrine duct implantation during surgery. Our case is a 66-year-old male patient who underwent an excision of a melanocytic neoplasm in which a reactive epithelial sheath neuroma was incidentally discovered in the excision specimen, adjacent to the biopsy site cicatrix. Histologically, there was benign cutaneous nerve hyperplasia with a proliferation of squamous epithelium in intimate apposition to the nerve bundles in the superficial dermis. We postulate that the process active in the formation of re-excision perineural invasion is the same as in epithelial sheath neuroma and that minor trauma not appreciable on histologic examination is responsible in the latter entity. We performed IL-6 staining and documented that IL-6 was upregulated at the interface of the nerve and reactive epithelium, but was absent in nerves distant from the site of surgery, suggesting that IL-6 may be essential to the lesion's development. The recognition of reactive epithelial sheath neuroma including the subcategory of re-excision perineural invasion is crucial for the dermatopathologist to prevent mislabeling this reactive entity as a perineural squamous cell carcinoma, which has clinical consequences for the patient such as wider re-excision and radiation treatment. Additionally, we have identified a potential pathophysiologic basis for this lesion.

Peripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair. Biotechnol. Bioeng. 2016;113: 2041-2053. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc., (© 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

Intraneural perineurioma is a benign tumor developed from the perineurium and responsible for localized nerve hypertrophy. This uncommon tumor is characterized by a proliferation of perineural cells with a "pseudo-onion bulb" pattern. We report a sciatic nerve intraneural perineurioma in a 39-year-old patient., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.

Patterns of invasion and stromal response are understudied in vulvar squamous cell carcinoma. The aim of this study was to explore whether histologic features such as an infiltrative pattern of invasion and fibromyxoid stromal response (FMX-SR) are meaningful prognostic factors. We reviewed 143 vulvar squamous cell carcinoma resections and correlated patterns of invasion and stromal response with patient age, ethnicity, depth of invasion, tumor size, perineural invasion (S100/AE1/3 stain), lymph node involvement (LNI), extranodal extension, margin status, pathologic stage, and recurrence. Univariate analyses of continuous variables were performed using t tests, whereas Pearson χ tests were used for categorical variables. Logistic regression analyses examined the relationship between histopathologic characteristics and clinical outcomes. There was a statistically significant association between infiltrative tumors and an FMX-SR in comparison with noninfiltrative tumors (P<0.001). Tumors with FMX-SR were significantly more deeply invasive (P=0.0025) and more likely to have LNI (P=0.0364), extranodal extension (P=0.0227), and perineural invasion (P=0.0011) compared with tumors without FMX-SR. For cases with negative surgical margins, the association between tumors with FMX-SR and LNI was significantly strengthened (odds ratio=4.73, P=0.0042), even after adjustments for age, race, and depth of invasion (odds ratio=4.34, P=0.0154). The presence of both FMX-SR and an infiltrative pattern of invasion in tumors with negative margins was significantly associated with LNI (P=0.0235) and recurrence (P=0.0124). These results suggest that interactions between nerve, tumor, and stromal cells play a role in tumor progression and represent additional prognostic factors that help stratify those patients at highest risk for LNI, extranodal extension, and recurrence.

Background: Nerve invasion is a specific type of tumor expansion and characteristic manifestation of pancreatic cancer (PC), with an incidence rate ranging from 50% to 100%. It is an important prognostic factor for pancreatic cancer, and its early detection is helpful in the management of the disease. This study was undertaken to analyze retrospectively the relationship between neural invasion and multiple clinicopathological features and to provide evidences for clinicians in the management of neural invasion in patients with PC., Methods: Formalin-fixed paraffin-embeded specimens of PC taken from 215 patients were examined for the presence of neural invasion under a light microscope. Analyzed was the relationship between neural invasion and multiple clinicopathological feature including preoperative fasting blood glucose level, amylase level, serum CA19-9 level, abdominal pain, lumbar and back pain, and the expressions of p53 and Ki67 in tumor tissues., Results: Preoperative fasting blood glucose level, serum CA19-9 level and p53 positive cells in cancer tissue were increased with the rise of pathological grade (P<0.05). These indices were significantly higher in patients with neural invasion than in those without (P<0.05). Further analysis revealed a positive correlation between p53 and Ki67 overexpression and lymphatic metastasis (P<0.05). Referred pain was positively correlated with neural invasion (P<0.05). Patients with PC perineural invasion were more likely to have a higher pathological grade (P<0.05)., Conclusions: Our data indicated that the preoperative fasting blood glucose level, serum CA19-9 level, and referred pain are novel predictive markers for neural invasion in patients with PC. p53 and Ki67 play important roles in neural invasion of PC. Management of hyperglycemia may serve as an auxiliary treatment to curb neural invasion in PC.

Physiological changes and pathological alterations in the nervous system of rodents are associated with modifications in the levels of neuroactive steroids in the brain, spinal cord and/or peripheral nerves. Measures of tissue levels of steroids in the nervous system present serious limitations for human studies and for longitudinal studies in animals. In this study we have explored whether levels of neuroactive steroids in plasma and the cerebrospinal fluid reflect their levels in neural tissues. To this aim, we have evaluated by liquid chromatography-tandem mass spectrometry the levels of several neuroactive steroids in plasma, cerebrospinal fluid, cerebral cortex, cerebellum, hippocampus, spinal cord and sciatic nerve of male and female rats. Data indicate that plasma and cerebrospinal fluid levels of steroids do not fully reflect their tissue levels. However, the interindividual variations in the levels of all the steroids assessed, with the exception of dehydroepiandrosterone, showed a positive correlation in plasma and cerebral cortex. Most steroids also showed a positive correlation in plasma and the cerebellum, the spinal cord and the sciatic nerve. In the hippocampus, the levels of tetrahydroprogesterone, testosterone and testosterone metabolites showed a significant positive correlation with their respective levels in plasma. The cerebrospinal fluid levels of some steroids, such as testosterone and dihydrotestosterone, showed a full correlation with tissue levels. In addition, cerebrospinal fluid levels of pregnenolone, progesterone, and 17β-estradiol showed a positive correlation with their corresponding levels in the majority of the neural structures analyzed. These findings suggest that the levels of some neuroactive steroids in cerebrospinal fluid as well as in plasma may be valuable to predict their levels in the nervous system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The objective was to assess the effect of topically administered insulin-like growth factor (IGF I) on peripheral nerve regeneration and functional recovery. Eighty male healthy white Wistar rats were divided into four experimental groups (n=20), randomly: in transected group (TC), the left sciatic nerve was transected and stumps were fixed in the adjacent muscle. In treatment group, defect was bridged using an inside-out artery graft (IOAG/IGF) filled with 10 μL IGF I (100 ng/kg). In artery graft group (IOAG), the graft was filled with phosphate-buffered saline alone. In sham-operated group (SHAM), sciatic nerve was exposed and manipulated. Each group was subdivided into five subgroups of five animals each and regenerated nerve fibres were studied 4, 8, 12 and 16 weeks after surgery. Behavioural testing, sciatic nerve functional study, gastrocnemius muscle mass and morphometric indices confirmed faster recovery of regenerated axons in IOAG/IGF than IOAG group (P<0.05). In immunohistochemistry, location of reactions to S-100 in IOAG/IGF was clearly more positive than that in IOAG group. When loaded in an artery graft, IGF I accelerated and improved functional recovery and morphometric indices of sciatic nerve., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The objective was to assess the effect of topically administered diclofenac on peripheral nerve regeneration and functional recovery. Eighty male healthy white Wistar rats were divided into four experimental groups (n = 20), randomly: In transected group (TC), left sciatic nerve was transected and stumps were fixed in the adjacent muscle. In treatment group defect was bridged using an artery graft (AG/DICLO) filled with 10 μL diclofenac (0.1 mg/kg). In artery graft group (AG), the graft was filled with phosphate-buffered saline alone. In sham-operated group (SHAM), sciatic nerve was exposed and manipulated. Each group was subdivided into four subgroups of five animals each and regenerated nerve fibers were studied 4, 8, 12 and 16 weeks after surgery. Behavioral testing, sciatic nerve functional study, gastrocnemius muscle mass and morphometric indices confirmed faster recovery of regenerated axons in AG/DICLO than AG group (p < 0.05). In immunohistochemistry, location of reactions to S-100 in AG/DICLO was clearly more positive than that in AG group. When loaded in an artery graft, diclofenac improved functional recovery and morphometric indices of sciatic nerve. Topical application of this readily available agent offers the benefit of cost savings as well as avoiding the complications associated with systemic administration., (Copyright © 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Notalgia paraesthetica is a neuropathic pruritus on the back. The aim of this retrospective study was to examine patient characteristics in a consecutive cohort from Brazil and Germany. A total of 65 patients (49 women, 16 men; age range 25-80 years, mean 56.2 ± 12.7 years; median 57.0 years) were investigated in order to determine the spinal or peripheral origin of notalgia paraesthetica. Protein gene product 9.5-positive intraepidermal nerve fibers were significantly reduced in the pruritic compared with the non-lesional area (p < 0.05). In 32.3% of patients, radiological examinations showed a stenosis and in 47.7% a degeneration. A correlation between the radiological findings and the exact dermatomal localization of notalgia paraesthetica was found in 15.7% of the involved areas. The significant reduction in intraepidermal nerve fiber density suggests that damage to the peripheral nerves is a more important aetiological factor than spinal changes in notalgia paraesthetica.

Liver transplantation (LT) reportedly prolongs the survival of patients with familial amyloidotic polyneuropathy (FAP), a fatal hereditary systemic amyloidosis caused by mutant transthyretin (TTR). However, what happens in systemic tissue sites long after LT is poorly understood. In the present study, we report pathological and biochemical findings for an FAP patient who underwent LT and died from refractory ventricular fibrillation more than 16&emsp14;years after FAP onset. Our autopsy study revealed that the distributions of amyloid deposits after LT were quite different from those in FAP amyloidogenic TTR V30M patients not having had LT and seemed to be similar to those observed in senile systemic amyloidosis (SSA), a sporadic systemic amyloidosis derived from wild-type (WT) TTR. Our biochemical examination also revealed that this patient's cardiac and tongue amyloid deposits derived mostly from WT TTR. We propose that FAP patients after LT may suffer from SSA-like WT TTR amyloidosis in systemic organs.

Background and Objectives: A critical challenge encountered in interventional pain medicine procedures is to accurately and efficiently identify transitions to peripheral nerve targets. Current methods, which include ultrasound guidance and nerve stimulation, are not perfect. In this pilot study, we investigated the feasibility of identifying tissue transitions encountered during insertions toward peripheral nerve targets using optical spectroscopy., Methods: Using a custom needle stylet with integrated optical fibers, ultrasound-guided insertions toward peripheral nerves were performed in 20 patients, with the stylet positioned in the cannula of a 20-gauge stimulation needle. Six different peripheral nerves were represented in the study, with 1 insertion per patient. During each insertion, optical reflectance spectra were acquired with the needle tip in subcutaneous fat, skeletal muscle, and at the nerve target region. Differences in the spectra were quantified with 2 parameters that provide contrast for lipid and hemoglobin, respectively., Results: The transition of the needle tip from subcutaneous fat to muscle was associated with lower lipid parameter values (P = 0.003) and higher hemoglobin parameter values (P = 0.023). The transition of the needle tip from the muscle to the nerve target region was associated with higher lipid parameter values (P = 0.008)., Conclusions: The results indicate that the spectroscopic information provided by the needle stylet could potentially allow for reliable identification of transitions from subcutaneous fat to skeletal muscle and from the muscle to the nerve target region during peripheral nerve blocks.

The extracellular matrix surrounds different neuronal compartments in the mature nervous system. In a variety of vertebrates, most brain regions are loaded with a distinct type of extracellular matrix around the somatodendritic part of neurons, termed perineuronal nets. The present study reports that chondrotin sulfate proteoglycan-based matrix is structured differently in the human lateral geniculate body. Using various chondrotin sulfate proteoglycan-based extracellular matrix antibodies, we show that perisomatic matrix labeling is rather weak or absent, whereas dendrites are contacted by axonal coats appearing as small, oval structures. Confocal laser scanning microscopy and electron microscopy demonstrated that these typical structures are associated with synaptic loci on dendrites. Using multiple labelings, we show that different chondrotin sulfate proteoglycan components of the extracellular matrix do not associate exclusively with neuronal structures but possibly associate with glial structures as well. Finally, we confirm and extend previous findings in primates that intensity differences of various extracellular matrix markers between magno- and parvocellular layers reflect functional segregation between these layers in the human lateral geniculate body., (Copyright © 2011 Wiley Periodicals, Inc.)

Objective: To explore a time-efficient method of identifying motor and sensory fascicles in peripheral nerve trunk., Methods: Thirty Wistar rats were selected to obtain whole spine. The spinal dorsal roots and ventral roots, and sciatic nerve were harvested as sensor, motor, and mixed samples, annexin V and agrin specificities were observed with Western blot and immunohistochemistry. A total of 32 New Zealand rabbits were selected and killed. The roots of spinal nerves were exposed under an operating microscope, and the ventral and dorsal roots, ∼3 mm to 5 mm, were dissociated, and frozen as transverse sections of 30-μm thickness. The sections were examined by micro-Raman spectroscopy., Results: The annexin V and agrin were special substances of sensory and motor nerves, respectively, and can act as specific antigens for identifying different nerve fascicles. Sections of the same type of nerve fascicles showed reproducibility with similar spectral features. Significant differences in the spectral properties, such as the intensity and breadth of the peak, were found between motor and sensory fascicles in the frequency regions of 1,088 cm(-1), 1,276 cm(-1), 1,439 cm(-1), 1,579 cm(-1), and 1,659 cm(-1). With the peak intensity ratio of 1.06 (I(1276)/I(1439)) as a standard, we could identify motor fascicles with a sensitivity of 88%, specificity of 94%, positive predictive value of 93%, and negative predictive value of 88%. In the range of 2,700 cm(-1) to 3,500 cm(-1), the half-peak width of the motor fascicles was narrow and sharp, whereas that of the sensory fascicles was relatively wider. A total of 91% of the peak features were in accordance with the identification standard., Conclusion: Motor and sensory fascicles exhibit different characteristics in Raman spectra, which are constant and reliable. Therefore, it is more effective than immunohistochemistry method in identifying different nerve fascicles according to the specific spectrum, and it possesses feasibility for clinical application.

The Merlin/NF2 tumor suppressor restrains cell growth and tumorigenesis by controlling contact-dependent inhibition of proliferation. We have identified a tight-junction-associated protein complex comprising Merlin, Angiomotin, Patj, and Pals1. We demonstrate that Angiomotin functions downstream of Merlin and upstream of Rich1, a small GTPase Activating Protein, as a positive regulator of Rac1. Merlin, through competitive binding to Angiomotin, releases Rich1 from the Angiomotin-inhibitory complex, allowing Rich1 to inactivate Rac1, ultimately leading to attenuation of Rac1 and Ras-MAPK pathways. Patient-derived Merlin mutants show diminished binding capacities to Angiomotin and are unable to dissociate Rich1 from Angiomotin or inhibit MAPK signaling. Depletion of Angiomotin in Nf2(-/-) Schwann cells attenuates the Ras-MAPK signaling pathway, impedes cellular proliferation in vitro and tumorigenesis in vivo., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pompe disease is caused by the deficiency of acid α-glucosidase (GAA), which degrades glycogen into glucose. Its manifestation is characterized by a broad and continuous spectrum of clinical severity ranging from severe infantile to relatively benign adult form. We describe a 12-year-old girl diagnosed at a presymptomatic stage of late form Pompe disease due to fortuitous detection of an elevated level of serum creatine kinase (CK) at the age of 4. Biopsies were taken from the quadriceps muscle and studied with histological and histochemical techniques, as well as in electron microscope. Sporadic muscle cells showed the accumulation of lysosomal glycogen, suggesting Pompe disease. Interestingly, we found lysosomal bound glycogen, located in the axons of intramuscular nerves. The diagnosis was confirmed by deficient GAA activity in leukocytes. Mutation analysis revealed changes IVS1-13T>G and p.C103G in the GAA gene. The patient was able to obtain enzyme replacement therapy in the early asymptomatic stage of the disease., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Unlabelled: In familial amyloid polyneuropathy (FAP), destruction of nerve fibres is related to accumulation of mutated transthyretin (mTTR) derived amyloid deposits (AD) in the endoneurium. Liver transplantation (LT), which removes the main source of mTTR, does not prevent deterioration of the clinical condition in all recipients., Material and Methods: We evaluated the distribution of AD in the central and peripheral nervous system in order to better understand the pathophysiology of FAP and the potential role of lesions of nerve blood vessels and of mTTR released by choroid plexuses (CP). Forty nerve biopsy specimens and 3 autopsy cases, including 7 patients who underwent liver transplantation, all from patients with symptomatic FAP and DNA mutation of the TTR gene, were included., Results: Patients were ranged into three categories: MORPHOLOGICAL CHANGES: Amyloid predominated around endoneurial capillaries in 37 patients, with occlusion/destruction of endoneurial capillaries in 15 nerves at late stages of the disease. Post-mortem examination showed amyloid in choroid plexuses and perivascular spaces in the brain and around blood vessels penetrating the endoneurium, following arachnoid and connective tissue septae. Destruction of endoneurial blood vessels is a late event in the natural course of FAP. Morphological findings were similar in patients who underwent liver transplantation and in those who did not. The distribution of amyloid in areas communicating with the subarachnoid space suggests that mutated TTR released in the CSF may move to the endoneurial fluid and accumulate in peripheral nerves, accounting for lack of efficacy of liver transplantation in some individuals.

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

Objective: We investigated neurotrophic activity of extracts from pre-degenerated and non-pre-degenerated peripheral nerves (complete extracts and extracts with fractions of narrower range of molecular weight) on the injured hippocampus., Methods: The experiment was carried out on male Wistar C rats. The complete extracts or fractions with different ranges of molecular weight were introduced to the site of injury with the autologous connective tissue chambers. We examined DiI-labeled hippocampal cell and AChE-positive nerve endings to assess the regeneration intensity., Results: The highest number of labeled hippocampal cells was observed in the group treated with fraction of molecular weight 10-100 kDa (72.5 +/- 13.7) obtained from pre-degenerated nerves. We observed the presence of AChE-positive fibers inside all examined chambers., Discussion: These results demonstrate that suitable modification of CNS environments by introducing the protein fractions obtained from peripheral nerves can initiate the regeneration of the damaged hippocampal structure in adult rats. Moreover, it is possible to intensify their neurotrophic effect by former pre-degeneration of peripheral nerves and extraction from the entire extract proteins of molecular weight of 10-100 kDa.

Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does not contain any motor fibres and Ia-afferents. On account of its virtually mixed fibre qualities, most researchers today recommend the cervical branch of the equine accessory nerve (AN) for harvesting diagnostic samples. Thus, the present study was carried out to gain morphometrical proof of the AN composition and to obtain stereological base values in healthy individuals using state-of-the-art technology. All parameters were compared to the common peroneal nerve (CPN), known to harbour all myelinated fibre classes. As this second biopsy site is located farther distally to the neuro-axis, attention was paid to possible length-dependent features. Taken together, digital image analysis could be accurately applied on all AN samples. Stereology supported the histological and clinical evidence that the AN contains all myelinated fibre types. The huge range and scatter of fibre counts and density (3351-17,812/mm(2)) per fascicle were comparable to that measured in the equine common peroneal, deep peroneal, lateral palmar and recurrent laryngeal nerves. Similar to those, fibre diameter distribution was bimodal with slow Abeta- and Agamma-mechanoceptor afferents outnumbering large myelinated Aalpha-fibres by a factor of about 1.5. With a g-ratio at 0.55 +/- 0.001, the overall degree of myelination in the AN is highly consistent and insignificantly ranges between that of the equine common peroneal and LPNs. Apart from this subtle deviation, a statistically relevant difference between the more proximal AN and the distal CPN could not be documented. By obtaining morphometrical standard parameters and even more sophisticated distribution indices, stereology is a valuable tool for detection of subtle changes that are likely to escape from the investigators' eyes. The AN serves as a reliable source for advanced peripheral nerve research and should be accompanied by farther distal nerve probes for assessment of neuropathies that present with a proximodistal gradient.

We report a simple electrochemical method for the identification of properties of peripheral nerve fibers, based on the detection of a neurotransmitter enzyme, acetylcholinesterase (AChE). A poly(diallydimethylammonium) (PDDA) adulterated poly(dimethylsiloxane) (PDMS) film is spin-coated on the surface of gold electrodes. Gold nanoparticles (AuNPs) are in situ synthesized on the polymer film, which act as "electron antennae" between the film and the electrode surface and also provide a biocompatible interface. This PDMS-PDDA/AuNPs film shows different adsorption sites to choline oxidase (ChO) and AChE; after incubation with ChO, the polymer-gold nanocomposite film also shows excellent adsorption ability to AChE. Moreover the adsorption sites of AChE would not be blocked by bovine serum albumin (BSA) which provides a good platform for the quantitative amperometric determination of AChE via the oxidation of the enzymatically generated H 2O 2 in the bienzyme system in the presence of acetylcholine. The detection limit is down to 1.0 unit/mL. The polymer-gold nanocomposite film shows excellent anti-interference ability to the coexistent electroactive substances such as ascorbic acid. Thus it was applied to determine AChE in peripheral nerve fibers homogenates and identify the motor and sensory fibers for the first time. Compared with histochemical staining methods, the electrochemical technique shows good accurate rate and faster response, which has good potential for a clinical application.

Peripheral nerves consist of 3 layers with differing characteristics: the endoneurium, perineurium, and epineurium. The perineurium represents a continuum with the pia-arachnoid from the central nervous system and extends distally with the sheath of capsular cells of peripheral sensorial organs and propioceptive receptors. It is made of layers of flattened cells surrounded by a basement membrane and collagen fibers, forming concentrically laminated structures around single nerve fascicles. Functionally, the perineurium modulates external stretching forces (that could be potentially harmful for nerve fibers), and along with endoneurial vessels, forms the blood-nerve barrier. Multiple pathologic conditions associated with the perineurium have been described. Perineurial invasion is considered an important prognostic factor in several malignant neoplasms. Perineuriomas are true benign infrequent perineurial cell neoplasms that have been divided in 2 categories: those with intraneural localization and a more common extraneural (soft tissue) group, including sclerosing and reticular variants. Sporadic cases of malignant perineuromas have been reported. Interestingly, neurofibromas and malignant peripheral nerve sheath tumors may also display perineurial cell differentiation. The histologic appearance of perineuriomas may overlap with other soft tissue spindle cell neoplasms. Immunohistochemistry is imperative for the diagnosis, although in certain cases ultrastructural studies may be needed. Typical perineuriomas are positive for epithelial membrane antigen, glucose transporter-1-1, and claudin-1, and negative for S-100 protein and neurofilaments. Perineuriomas have mostly simple karyotypes, with one or few chromosomal rearrangements or numerical changes and it seems that specific cytogenetic aberrations may correlate with perineurioma subtype.

The dorsal vessel of the Vietnamese stick insect, Baculum extradentatum, consists of a tubular heart and an aorta that extends anteriorly into the head. Alary muscles, associated with the heart, are anchored to the body wall with attachments to the dorsal diaphragm. Alary muscle contraction draws haemolymph into the heart through incurrent ostia. Excurrent ostia lie on the dorsal vessel in the last thoracic and in each of the first two abdominal segments. Muscle fibers are associated with these excurrent ostia. Crustacean cardioactive peptide (CCAP)- and proctolin-like immunoreactivity is present in axons of the segmental nerves that project to the dorsal vessel, and in processes extending over the heart and alary muscles. Proctolin-like immunoreactive processes are also localized to the valves of the incurrent ostia and to the excurrent ostia. Neither the link nerve neurons, nor the lateral cardiac neurons, stain positively for these peptides. Physiological assays reveal dose-dependent increases in heart beat frequency in response to CCAP and proctolin. Isolating the dorsal vessel from the ventral nerve cord led to a change in the pattern of heart contractions, from a tonic, stable heart beat, to one which was phasic. The tonic nature was restored by the application of CCAP.

Newly hatched paralarvae ("hatchlings") or late-stage embryos of Loligo opalescens were dissected and pieces of tissue removed for immunostaining as flat whole mounts. The general layout of the peripheral nervous system in the mantle and gills was investigated using antisera for tubulin and FMRFamide. Primary sensory neurons are densely distributed in the outer mantle epidermis and show strong FMRFamide immunoreactivity. Their axons form a plexus in the underlying dermis, but do not appear to innervate the chromatophore muscles, which are well visualized with anti-tubulin. Some cross the muscle layer and enter the stellate ganglia via the stellar nerves. The stellate ganglion neuropil contains a rich FMRFamide-immunoreactive mass of axons. It is suggested that these axons originate in large part from sensory neurons in the skin and that the known modulatory effects of FMRFamide-related peptides on motor output of the stellate ganglion may be a reflection of this sensory input in normal life. FMRFamide-immunoreactive primary sensory neurons are also abundant in the gills, but unlike those in the mantle, these cells lack cilia or other external projections. Anti-tubulin staining reveals a network of interstitial cells in the mantle dermis. Such networks may have been mistaken for nerve nets in older accounts. Additional results with Octopus vulgaris hatchlings and immunostaining for serotonin (5HT), small cardioactive peptide (SCP), and gonadotropin releasing hormone (GnRH) are briefly reported.

Objective: To explore a method to identify the sensory and motor fascicles in peripheral nerve trunk., Methods: Thirty Wistar rats were selected to obtain whole spine. The spinal ganglion, its dorsal root and ventral root, and sciatic nerve were harvested, Annexin V and Agrin specificities were observed with Western blot. In the experimental group, anterior branch and posterior branch of spinal nerve, sciatic nerve, and its muscular branch and cutaneous branch were harvested from 15 rats to make the observation of immunohistochemistry. In the other 15 rats, first antibody was replaced by PBS as control group. Different nerve fascicles were studied with Micro Raman scattering technique in 16 12-month-old New Zealand rabbits., Results: The Annexin V and Agrin were special substances of sensory and motor nerves respectively and can act as specific antigens for identifying different nerve fascicles. There were significant differences in the intensity and breadth of the peak of the spectral properties between motor and sensory fascicles at frequencies of 1,088, 1,276, 1,439, 1,579 and 1,659 cm(-1). The peak intensity ratios of 1,276 to 1,439 cm(-1) were 0.95+/-0.06 in motor nerve fascicles and 1.17+/-0.08 in sensory fascicles, showing significant differences (P<0.05)., Conclusion: The Micro Raman spectra is more effective than immunohistochemistry in identifying different nerve fascicles, and it possesses as feasibility for clinical application.

A large number of genes regulate the process of regeneration after the nerve injury. Facial nerve axotomy of rats provides us with a useful model in which to study the molecular and cellular mechanisms of peripheral nerve regeneration. We performed the subtracted library screening of the facial nucleus after the facial nerve axotomy. We identified the sonic hedgehog (Shh) as a candidate molecule that regulates regeneration of injured neurons. By making adenoviral vector that transfers Shh, we found that the motor neuron survival was significantly promoted for 3-5 days after neonate rat injury. Furthermore, the continuous application of cyclopamin, which is a signal inhibitor of Smoothened, to the injured site, motor neuron death was induced in the adult rats after axotomy. These results indicate that Shh may play a regulatory role in the regeneration after nerve injury.

The prevalence of motor variations in the nerves supplying muscles of the first web space was evaluated by a visual dissection and immunohistochemical analysis from 56 cadaver hands. By microscopic visualization, 30% of the superficial radial nerves (SRNs) sent branches into muscles of the first web space. Since these unexpected penetrating branches were expected to be sensory or proprioceptive, markers of sensory and motor axons were used for confirmation. Positive identifications of motor axons (as identified by positive immunostaining for choline acetyltransferase) were made in 30% of SRNs and in 28.5% of posterior interosseous nerves. Classical teachings that the SRNs and PINs are exclusively sensory have been brought into question. Our data are in agreement with the rare clinical finding that motor function occasionally persists following devastating injury to both the ulnar and median nerves. Anatomic prevalence for this variation appears much higher than previous descriptions have indicated.

With the use of increasingly sensitive methods for detection of the abnormal isoform of prion protein (PrP(Sc)) and infectivity in prion diseases, it has recently been shown that parts of the peripheral nervous system (PNS) of bovine spongiform encephalopathy (BSE)-affected cattle may become infected. It has been reported that prions spread to the central nervous system (CNS) via the PNS in sheep scrapie, but the pathogenesis of BSE in cattle is less well understood. To determine whether parts of the PNS other than those implicated directly in the hypothetical pathogenetic spread of agent from the intestine to the CNS become involved before or after the CNS is affected, PrP(Sc) distribution was investigated by a highly sensitive Western blotting technique in dorsal root ganglia, stellate ganglion, phrenic, radial and sciatic nerves, adrenal gland and CNS of cattle that were inoculated orally with BSE-affected brain and culled sequentially. In experimentally BSE-affected cattle, PrP(Sc) was first detected in the CNS and dorsal root ganglia; subsequently, PrP(Sc) accumulation was detected in the peripheral nerve trunks. PrP(Sc) was also detected in the adrenal glands of cattle that showed clinical signs. No PrP(Sc) was detected in the PNS of BSE-negative cattle. This study shows that, with respect to dorsal root ganglia, a paravertebral sympathetic ganglion and the somatic nerves examined, PrP(Sc) is detected in the PNS during the disease course at the same time as, or after, it accumulates in the CNS.

For proteomic analysis, sample preparation plays a crucial role in two-dimensional gel electrophoresis (2DE), since, very often, each tissue or cell culture requires specific treatments. In the present paper, we report a sample preparation procedure suitable for 2DE that was done on peripheral nerve using bovine sciatic nerves and human sural nerve biopsies. We obtained an appreciable reduction of tissue heterogeneity using protein extracts obtained from nerve-fiber bundles instead of the entire nerve. In addition, we optimized 2DE protein separation using a combination of CHAPS, Triton X-100, and SB3-10 detergents in an isoelectric-focusing (IEF) buffer. The reported experimental procedures appear to be essential for 2DE separation of peripheral nerve proteins for the establishment of a reference map.

Three 80- to 95-month-old Holstein dairy cattle infected naturally with the agent of bovine spongiform encephalopathy (BSE) and slaughtered at abattoirs in Japan were examined for the distribution of disease-specific and protease-resistant prion protein (PrP(Sc)) by immunohistochemistry (IHC) and Western blot (WB) analyses. The cattle showed no clinical signs or symptoms relevant to BSE but were screened as positive by enzyme-linked immunosorbent assay, a rapid test for BSE. This positive result was confirmed by IHC or WB in a specimen of the medulla oblongata. Histopathologically, these cattle showed no vacuolation in tissue sections from the central nervous system except for the medulla oblongata. Both IHC and WB analyses revealed PrP(Sc) accumulation in the brain, spinal cord, satellite and ganglionic cells of the dorsal root ganglia, and the myenteric plexus of the distal ileum. In addition, small amounts of PrP(Sc) were detected in the peripheral nerves of 2 cattle by WB. No PrP(Sc) was demonstrated by either method in the Peyer's patches of the distal ileum; lymphoid tissues including the palatine tonsils, lymph nodes, and spleen; or other tissues. The distribution of PrP(Sc) accumulation in the preclinical stage was different between naturally infected cattle and cattle inoculated experimentally with the BSE agent.

The endoneurial extracellular matrix (ECM) molecules are involved in cell signalling during nervous system development and regeneration. Quantitative differences of immunofluorescence labelling for chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), tenascin-C (TN-C), and thrombospondin (TSP) were evaluated in intact rat dorsal and ventral roots and dorsal and ventral roots 2 and 4 weeks after rhizotomy using image analysis. The distal stumps of spinal roots displayed increased immunolabelling for the molecules with higher immunofluorescence in dorsal than in ventral roots up to 2 weeks from transection. Four weeks after rhizotomy, the immunoreactivity for CSPG, TN-C and TSP decreased in dorsal and increased in ventral root stumps, although a higher level of immunofluorescence for FN remained in both dorsal and ventral root stumps 4 weeks after injury in comparison to 2 weeks after injury. We suggest that the amount of some ECM molecules changed differentially 2 and 4 weeks after rhizotomy to create an appropriate environment in the endoneurium for early and later regrowth of sensory and motor axons. The results presented here are the first report of differences between the endoneurial ECM content of damaged afferent and motor nerve fibers. In addition, the immunohistochemical detection of individual ECM molecules indicated that final extrinsic conditions stimulating the regrowth of regenerating axons probably arise from a balance of both growth-promoting and -inhibiting molecules in the endoneurium.

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Peripheral nerves provide essential connections between the central nervous system and muscles, autonomic structures and sensory organs. Nitric oxide (NO) participates in critical actions involving several aspects of peripheral nerve function and disease. It offers important roles in "normal" afferent signaling of pain through the dorsal horn of the spinal cord and in autonomic control through nitrergic innervation. NO is generated during the fundamental processes of Wallerian degeneration of peripheral nerves following injury that bear on subsequent regenerative events. Through its actions on vasa nervorum, the blood supply to nerves, NO participates in microvascular changes following injury but also has direct roles in axon and myelin breakdown and "clearance" prior to regeneration. During such processes, NO contributes to the development of neuropathic pain. Excessive local levels of NO during inflammation may damage axons and growth cones. Low-grade chronic rises in NO may also contribute toward peripheral nerve damage, or neuropathy in diabetes. In this review, we consider the evidence for these roles and their potential importance in disease and repair of peripheral nerves.

The authors describe a simple and cost-effective method of immunostaining semithin epoxy resin sections of peripheral nerve for light microscopy with antibodies to myelin protein zero, peripheral myelin protein 22, myelin basic protein, and neurofilament protein 200 using a combined technique of surface etching with sodium ethoxide and heat-induced antigen retrieval.

In order to clarify the role of angiogenic factors in polyneuropathy of POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome, we measured the serum concentrations of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in 11 patients and correlated these with VEGF and EPO peripheral nerve expression and the degree of endoneurial vessel involvement. We found that POEMS syndrome was associated with high levels of serum VEGF and, conversely, low levels of serum EPO. Similarly, in POEMS nerves VEGF was highly expressed in blood vessels and some non-myelin-forming Schwann cells. In contrast, the expression of VEGF receptor 2 was down-regulated compared with that in normal nerves. Both EPO and EPO receptor were localized to the nerve vasculature and were expressed to similar extents in normal and POEMS nerves. The inverse correlation between VEGF and EPO serum levels was maintained during the clinical course; however, both levels returned to normal when there was a response to therapy. High serum VEGF, low serum EPO and high peripheral nerve VEGF were all associated with more severe endoneurial vessel involvement and nerve damage. Light microscopy showed an increased thickness of the basal lamina and a narrowing of the lumina of endoneurial vessels in POEMS samples, while proliferation of endothelial cells and opening of tight junctions were observed by electron microscopy. The present data support the role of angiogenic factors as diagnostic and prognostic markers of POEMS syndrome. They also suggest that VEGF and EPO are involved in the pathogenesis of polyneuropathy. In conclusion, establishing the role of angiogenic factors in polyneuropathy may lead to a better understanding of the effects of VEGF and EPO on microangiopathy and Schwann cell function.

Aims: Perineural invasion (PNI) is one of the few unequivocal criteria to diagnose adenocarcinoma of the prostate. Distinguishing PNI from perineural indentation (PNIn), however, is sometimes difficult. The aim of this study was to discriminate between PNI and PNIn using EMA immunohistochemistry., Methods and Results: We selected representative sections from 87 prostatectomies with prostatic adenocarcinoma. Normal peripheral nerves were continuously encircled with perineurium, which was immunoreactive for EMA. We identified 1319 PNI by carcinomas, 368 PNIn by carcinomas, and 303 PNIn by benign glands. We categorized the EMA immunoreactivity patterns into three classes: samples that displayed discontinuity or complete loss of the perineurium (Type A), samples where there were carcinomas or benign glands in the perineural space or peripheral nerves (Type B) and samples that showed no changes in the perineurium (Type C). For PNI we observed Type A, Type B, and Type C patterns in 55.3%, 24.8% and 19.9% of carcinomas, respectively. The incidence of each of those patterns in PNIn by carcinomas was 32.1%, 14.9% and 53.0%, respectively. Cases of PNIn by benign glands showed Type A or Type C patterns. They did not, however, exhibit Type B patterns., Conclusion: EMA immunostaining will aid the diagnosis of prostatic adenocarcinoma.

We present three cases of subcutaneous tumors with hybrid features of schwannoma-perineurioma (one case) and neurofibroma-perineurioma (two cases), which occurred in two women aged 50 and 52 years and one man aged 52. Locations included the scapular area, skin overlying breast and knee area. The tumors were 1.5, 4 and 5 cm in largest diameter. None of the patients had signs of neurofibromatosis. All tumors were surgically removed, and patients remained disease-free for 1 to 4 years. The classification of the lesion into schwannoma-perineurioma and neurofibroma-perineurioma rested on histopathological and immunohistochemical findings. An ultrastructural study was performed in one case of neurofibroma-perineurioma. All cases were studied for mutation of the NF2 gene, and in one case (neurofibroma-perineurioma) a point mutation was detected in exon 15 of the gene.

In order to investigate whether normal myelinated primary afferent axons sprout into the territories of adjacent injured peripheral nerve fibers in the superficial dorsal horn of the spinal cord, adult rats underwent either sectioning of the saphenous or femoral nerves on one side, or else unilateral denervation of the skin of the posterior thigh. Two weeks later cholera toxin B subunit (CTb), which is normally transported selectively by myelinated somatic primary afferents, was injected into the ipsilateral (intact) sciatic nerve. The relationship between CTb, vasoactive intestinal peptide (VIP), and binding of Bandeiraea simplicifolia isolectin B4 (IB4) was then examined in the ipsilateral dorsal horn of the second to fifth lumbar spinal segments (L2-L5). Sectioning of the femoral or saphenous nerves resulted in a reduction of IB4 binding in laminae I-II in the medial third of the dorsal horn of L2, L3, and the upper part of L4. VIP-immunoreactivity was upregulated in exactly the same regions in which IB4-binding was reduced. These correspond to the areas that were previously innervated by unmyelinated afferents in the sectioned nerves. CTb-labeling was detected in regions known to receive input from myelinated sciatic afferents: lamina I and a band extending from the inner part of lamina II (IIi) to lamina V in the L3-5 segments, and the deepest part of the dorsal horn in L2. Importantly, no CTb-labeling was detected in the outer part of lamina II (IIo) in the denervated areas. Sectioning of branches of the posterior cutaneous nerve of the thigh resulted in a reduction of IB4-binding and upregulation of VIP-immunoreactivity in the lateral part of the superficial dorsal horn of caudal L4 and L5. Again, CTb-immunoreactivity showed the normal sciatic pattern in L4-L5, with no labeling detected in lamina IIo in the denervated region. These results do not support the suggestion that the central terminals of intact myelinated afferents sprout into regions of lamina II occupied by adjacent nerves that have been axotomized peripherally., (Copyright 2004 Wiley-Liss, Inc.)

Background: The disease-associated form of prion protein (PrP(res)) has been noted in lymphoreticular tissues in patients with variant Creutzfeldt-Jakob disease (vCJD). Thus, the disease could be transmitted iatrogenically by surgery or use of blood products. We aimed to assess transmissibility of the bovine spongiform encephalopathy (BSE) agent to primates by the intravenous route and study its tissue distribution compared with infection by the oral route., Methods: Cynomolgus macaques were infected either intravenously or orally with brain homogenates from first-passage animals with BSE. They were clinically monitored for occurrence of neurological signs and killed humanely at the terminal stage of the disease. Brain, lymphoreticular tissues, digestive tract, and peripheral nerves were obtained and analysed by sandwich ELISA and immunohistochemistry for quantitative and qualitative assessment of their PrP(res) content., Findings: Incubation periods after intravenous transmission of BSE were much shorter than after oral infection. We noted that PrP(res) was present in lymphoreticular tissues such as spleen and tonsils and in the entire gut from the duodenum to the rectum. In the gut, PrP(res) was present in Peyer's patches and in the enteric nervous system and nerve fibres of intestinal mucosa. Furthermore, PrP(res) was found in locomotor peripheral nerves and the autonomic nervous system. Amount of PrP(res) ranged from 0.02% to more than 10% of that recorded in brain. Distribution of PrP(res) was similar in animals infected by the intravenous or oral route., Interpretation: Our findings suggest that the possible risk of vCJD linked to endoscopic procedures might be currently underestimated. Human iatrogenic vCJD cases infected intravenously raise the same public-health concerns as primary cases and need the same precautionary measures with respect to blood and tissue donations and surgical procedures.

Dendritic cell neurofibroma with pseudorosettes (DCNWPR) is a recently proposed variant of neurofibroma. However, its peripheral nerve sheath origin has subsequently been questioned, and it has been suggested that the neoplasm could represent a hitherto undescribed variant of melanocytic nevus with neural differentiation. Here we report a case of DCNWPR that arose almost exclusively within the confinement of the perineurium in the skin. This observation gives further evidence that this entity is a peripheral nerve sheath tumor and is unrelated to melanocytic neoplasms.

Symbolism that describes the synthetic processes for chemical compounds has been used to describe, in qualitative terms, the synthesis of tissues and organs at the correct anatomical site. The synthetic process is summarized in the reaction diagram, a shorthand representation of the reactants, reactor, and products. Analysis of a large number of independent protocols has led to identification of the simplest synthetic pathways for two organs that have been studied extensively: skin and peripheral nerves. These apparently irreducible reaction diagrams for the two organs are not only simple but surprisingly similar, a fact suggesting the existence of general rules for the synthesis of other organs as well. The only two reactants that are required are an active scaffold (a macromolecular network synthesized as a highly porous analogue of the extracellular matrix) and a seeding of epithelial cells of the organ being synthesized. Scaffolds possessed biological (regenerative) activity provided that they were capable of blocking the contraction process that leads to closure of the injured site. Such activity requires that the density of ligands for binding of contractile cells on the scaffold maintains a sufficiently high level over the period of synthesis.

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16-fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.