Anti-neurofilament Antibody Research Articles

Neuromuscular Junction Development Differs Between Extraocular and Skeletal Muscles and Between Different Extraocular Muscles.

To determine whether development of neuromuscular junctions (NMJs) differs between extraocular muscles (EOMs) and other skeletal muscles. Mouse EOMs, diaphragm, and tibialis anterior (TA) were collected at postnatal day (P)0, P3, P7, P10, P14, and P21, and 12 weeks. Whole muscles were stained with α-bungarotoxin, anti-neurofilament antibody, and slow or fast myosin heavy chain antibody, and imaged with a confocal microscope. Images were quantified using Imaris software. NMJs in the EOMs show a unique pattern of morphological development compared to diaphragm and TA. At P0, diaphragm and TA NMJs were oval plaques; EOM single NMJs were long, thin rods. NMJs in the three muscle types progress to mature morphology at different rates. At all ages, EOM single NMJs were larger, especially relative to myofiber size. The inferior oblique and inferior rectus muscles show delayed single NMJ development compared to other EOMs. NMJs on multiply-innervated fibers in the EOMs vary widely in size, and there were no consistent differences between muscles or over time. Incoming motor nerves formed complex branching patterns, dividing first into superficial and deep branches, each of which branched extensively over the full width of the muscle. Motor axons that innervate multiply-innervated fibers entered the muscle with the axons that innervate singly-innervated fibers, then extended both proximally and distally. EOM NMJs had more subsynaptic nuclei than skeletal muscle NMJs throughout development. EOMs show a unique pattern of NMJ development and have more subsynaptic nuclei than other muscles, which may contribute to the exquisite control of eye movements.

Microsurgical and fiber tract anatomy of the interthalamic adhesion.

The authors of this study aimed to define the microanatomy of the interthalamic adhesion (ITA) using microfiber dissection, magnetic resonance (MR) tractography, and histological analysis. Sagittal, coronal, and axial MR images from 160 healthy individuals 2-82 years of age were examined. The relationships between age range and ITA morphology as well as between gender and ITA morphology were evaluated statistically. Among these 160 individuals, 100 who had undergone MR tractography were examined. In this group, the presence of fiber tracts in the ITA and the relationship with ITA morphological types were examined. Thirty formalin-fixed human cadaveric brains were also examined endoscopically, and 6 hemispheres were dissected from the medial to lateral and superior to inferior directions under the microscope. Sections taken from one of the brains with an ITA type 2 with both thalami were examined histologically. Anti-neurofilament antibody was used in the histological examination. Four morphological types of ITA were observed. Type 1 had an adhesion/adherent appearance, type 2 had a bridge/commissure appearance, type 3 showed no adhesion, and type 4 had a double bridge. Tractographic examination revealed that 28% had no fiber tract transition in the ITA, 21% had a significant transition, and 51% had an indistinct transition. Statistically, the presence of the ITA was significantly higher in the pediatric (age) and female (gender) groups. In specimens with ITAs of a bridge/commissure appearance (type 2), fiber tracts showed clear transitions between thalami. In type 1 (adherent/adhesive appearance), fiber tracts were observed within the ITA, but a reciprocal transition was unclear. Dissection showed that these fiber tracts in the ITA reach the nucleus accumbens, caudate nucleus, and frontoorbital region anteriorly and the lateral habenula and posterior commissure posteriorly. Some fibers also joined the ansa peduncularis. In histological studies, axonal fibers moving in the ITA were observed with anti-neurofilament antibody staining. This is the first study to demonstrate fiber tracts of the ITA through fiber dissection and transillumination techniques as well as radiological and histological study. Statistical data were obtained by comparing the morphological group with age and gender groups. The anatomy of this structure, which has been neglected for many years, was reexamined. This study showed that the ITA has fibers connecting different parts of the brain, in contrast to previous studies suggesting that it was a simple massa.

Distribution of Neurofilament in Peeled Internal Limiting Membrane.

The aim was to study the distribution of neurofilament in peeled internal limiting membrane (ILM). Prospective case study. Vitrectomy and ILM peeling were performed in patients with epiretinal membrane and macular hole. ILM flap specimens were obtained as one disc area size from five locations. Immunofluorescent staining was performed with an antineurofilament heavy antibody. Using a confocal microscope, retinal cell debris density was studied using the ImageJ program. Percent of stained neurofilament was 1.58 ± 1.14% in total (2.45 ± 1.37% in extranasal, 1.97 ± 0.75% in extratemporal, 1.93 ± 1.26% in juxta-nasal, 0.89 ± 0.69% in fovea, and 0.63 ± 0.46% in juxtatemporal). The Kruskal-Wallis test revealed significant differences among groups (P < 0.05). Bonferroni post hoc analysis only confirmed significant difference between juxtatemporal and extranasal groups (P < 0.05). In peeled ILM flap, neurofilaments are rarely detected in the juxtatemporal area. However, they are frequently detected in the extranasal area. [Ophthalmic Surg Lasers Imaging Retina 2023;54:643-648.].

Repeated intravenous infusion of mesenchymal stem cells for enhanced functional recovery in a rat model of chronic cerebral ischemia.

Stroke is a major cause of long-term disability, and there are few effective treatments that improve function in patients during the chronic phase of stroke. Previous research has shown that single systemic infusion of mesenchymal stem cells (MSCs) improves motor function in acute and chronic cerebral ischemia models in rats. A possible mechanism that could explain such an event includes the enhanced neural connections between cerebral hemispheres that contribute to therapeutic effects. In the present study, repeated infusions (3 times at weekly intervals) of MSCs were administered in a rat model of chronic stroke to determine if multiple dosing facilitated plasticity in neural connections. The authors induced middle cerebral artery occlusion (MCAO) in rats and, 8 weeks thereafter, used them as a chronic stroke model. The rats with MCAO were randomized and intravenously infused with vehicle only (vehicle group); with MSCs at week 8 (single administration: MSC-1 group); or with MSCs at weeks 8, 9, and 10 (3 times, repeated administration: MSC-3 group) via femoral veins. Ischemic lesion volume and behavioral performance were examined. Fifteen weeks after induction of MCAO, the thickness of the corpus callosum (CC) was determined using Nissl staining. Immunohistochemical analysis of the CC was performed using anti-neurofilament antibody. Interhemispheric connections through the CC were assessed ex vivo by diffusion tensor imaging. Motor recovery was better in the MSC-3 group than in the MSC-1 group. In each group, there was no change in the ischemic volume before and after infusion. However, both thickness and optical density of neurofilament staining in the CC were greater in the MSC-3 group, followed by the MSC-1 group, and then the vehicle group. The increased thickness and optical density of neurofilament in the CC correlated with motor function at 15 weeks following induction of MCAO. Preserved neural tracts that ran through interhemispheric connections via the CC were also more extensive in the MSC-3 group, followed by the MSC-1 group and then the vehicle group, as observed ex vivo using diffusion tensor imaging. These results indicate that repeated systemic administration of MSCs over 3 weeks resulted in greater functional improvement as compared to single administration and/or vehicle infusion. In addition, administration of MSCs is associated with promotion of interhemispheric connectivity through the CC in the chronic phase of cerebral infarction.

Automatic three-dimensional reconstruction of fascicles in peripheral nerves from histological images.

Computational studies can be used to support the development of peripheral nerve interfaces, but currently use simplified models of nerve anatomy, which may impact the applicability of simulation results. To better quantify and model neural anatomy across the population, we have developed an algorithm to automatically reconstruct accurate peripheral nerve models from histological cross-sections. We acquired serial median nerve cross-sections from human cadaveric samples, staining one set with hematoxylin and eosin (H&E) and the other using immunohistochemistry (IHC) with anti-neurofilament antibody. We developed a four-step processing pipeline involving registration, fascicle detection, segmentation, and reconstruction. We compared the output of each step to manual ground truths, and additionally compared the final models to commonly used extrusions, via intersection-over-union (IOU). Fascicle detection and segmentation required the use of a neural network and active contours in H&E-stained images, but only simple image processing methods for IHC-stained images. Reconstruction achieved an IOU of 0.42±0.07 for H&E and 0.37±0.16 for IHC images, with errors partially attributable to global misalignment at the registration step, rather than poor reconstruction. This work provides a quantitative baseline for fully automatic construction of peripheral nerve models. Our models provided fascicular shape and branching information that would be lost via extrusion.

Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method.

.Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.

Developmental studies on the acquisition of perception conducting pathways via TRP channels in rat molar odontoblasts using immunohistochemistry and RT-qPCR

Odontoblasts act as dentin formation and sensory receptors. Recently, it was reported that transient receptor potential ankyrin (TRPA) 1, TRP vanilloid (TRPV) 4 and pannexin 1 (PANX-1) play important roles in odontoblast sensory reception. However, it is not known when odontoblasts begin to possess a sense reception function. The aim of this study was to clarify the development of odontoblasts as sense receptors. Sections of mandibular first molars from postnatal day (PN) 0 to PN12 Wistar rats were prepared for hematoxylin–eosin staining. Immunohistochemically, we used anti-dentin sialoprotein (DSP), anti-TRPA1, anti-TRPV4, anti-PANX-1, and anti-neurofilament (NF) antibodies. In addition, we investigated TRPA1 and TRPV4 expression by reverse transcriptional quantitative polymerase chain reaction (RT-qPCR). At PN0, undifferentiated odontoblasts showed no immunoreaction to anti-DSP, anti-TRPA1, anti-TRPV4, or anti-PANX-1 antibodies. However, immunopositive reactions of these antibodies increased during odontoblast differentiation at PN3 and PN6. An immunopositive reaction of the anti-NF antibody appeared in the odontoblast neighborhood at PN12, when the odontoblasts began to form root dentin, and this appeared later than that of the other antibodies. By RT-qPCR, expression of TRPA1 at PN6 was significantly lower than that at PN0 (p < 0.05) and PN3 (p < 0.01). Expression of TRPV4 at PN6 was significantly lower than that at PN0 (p < 0.01) and PN3 (p < 0.01). The results of this study suggest that odontoblasts may acquire sensory receptor function after beginning to form root dentin, when TRPA1, TRPV4, PANX-1 channels, and nerve fibers are completely formed.

RGC Neuroprotection Following Optic Nerve Trauma Mediated By Intranasal Delivery of Amnion Cell Secretome.

PurposeIntranasally delivered ST266, the biological, proteinaceous secretome of amnion-derived multipotent progenitor cells, reduces retinal ganglion cell (RGC) loss, optic nerve inflammation, and demyelination in experimental optic neuritis. This unique therapy and novel administration route delivers numerous cytokines and growth factors to the eye and optic nerve, suggesting a potential to also treat other optic neuropathies. Thus, ST266-mediated neuroprotection was examined following traumatic optic nerve injury.MethodsOptic nerve crush injury was surgically induced in C57BL/6J mice. Mice were treated daily with intranasal PBS or ST266. RGC function was assessed by optokinetic responses (OKRs), RGCs were counted, and optic nerve sections were stained with luxol fast blue and anti-neurofilament antibodies to assess myelin and RGC axon damage.ResultsIntranasal ST266 administered daily for 5 days, beginning at the time that a 1-second optic nerve crush was performed, significantly attenuated OKR decreases. Furthermore, ST266 treatment reduced damage to RGC axons and myelin within optic nerves, and blocked RGC loss. Following a 4-second optic nerve crush, intranasal ST266 increased RGC survival and showed a trend toward reduced RGC axon and myelin damage. Ten days following optic nerve crush, ST266 prevented myelin damage, while also inducing a trend toward increased RGC survival and visual function.ConclusionsST266 significantly attenuates traumatic optic neuropathy. Neuroprotective effects of this unique combination of biologic molecules observed here and previously in optic neuritis suggest potential broad application for preventing neuronal damage in multiple optic nerve disorders. Furthermore, results support intranasal delivery as a novel, noninvasive therapeutic modality for eyes and optic nerves.

Preservation of Cells of the Organ of Corti and Innervating Dendritic Processes Following Cochlear Implantation in the Human: An Immunohistochemical Study.

This study evaluates the degree of preservation of hair cells, supporting cells, and innervating dendritic processes after cochlear implantation in the human using immunohistochemical methods. Surgical insertion of a cochlear implant electrode induces various pathologic changes within the cochlea including insertional trauma, foreign body response, inflammation, fibrosis, and neo-osteogenesis. These changes may result in loss of residual acoustic hearing, adversely affecting the use of hybrid implants, and may result in loss of putative precursor cells, limiting the success of future regenerative protocols. Twenty-eight celloidin-embedded temporal bones from 14 patients with bilateral severe to profound sensorineural hearing loss and unilateral cochlear implants were studied. Two sections including the modiolus or basal turn from each temporal bone were stained using antineurofilament, antimyosin-VIIa, and antitubulin antibodies in both the implanted and unimplanted ears. Inner and outer hair cells: Immunoreactivity was reduced throughout the implanted cochlea and in the unimplanted cochlea with the exception of the apical turn.Dendritic processes in the osseous spiral lamina: Immunoreactivity was significantly less along the electrode of the implanted cochlea than in the other segments.Inner and outer pillars, inner and outer spiral bundles, and Deiters' cells: Immunoreactivity was similar in the implanted and unimplanted cochleae. Insertion of a cochlear implant electrode may significantly affect the inner and outer hair cells both along and apical to the electrode, and dendritic processes in the osseous spiral lamina along the electrode. There was less effect on pillar cells, Deiters' cells, and spiral bundles.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus.

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3'-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Spinal nerve defects in mouse embryos prenatally exposed to valproic acid.

To examine in detail spinal nerve defects induced by prenatal exposure to valproic acid in mice, pregnant ICR mice were subcutaneously injected with a single dose of 400mg/kg valproic acid on gestational day 6, 7, 8, or 9, and their embryos were observed on gestational day 10. The whole-mount immunostaining using an anti-neurofilament antibody allowed us to identify spinal nerve defects, such as a loss of bundle, anastomosis among bundles arising from adjacent segment, and a disrupted segmental pattern of the dorsal root ganglia, in valproic acid-exposed embryos. The prevalence of spinal nerve defects was the highest in the embryos exposed to valproic acid on gestational day 8 among the experimental groups. Then, effects of the administration dose of valproic acid on the prevalence of spinal nerve defects were examined on gestational day 10 and found to be dose-dependently increased. It was noteworthy that all embryos exposed to 600mg/kg of valproic acid on gestational day 8 suffered spinal nerve defects. Folic acid (3mg/kg/day) supplementation during gestational day 6-10 suppressed the prevalence of valproic acid-induced neural tube defects, which are common malformations in offspring prenatally exposed to valproic acid, but not that of spinal nerve defects. Thus, the spinal nerve defects due to prenatal valproic acid exposure might be induced by mechanisms different from those of neural tube defects. Because spinal nerve defects were predicted to be caused by the disrupted segmental arrangement of the somites and/or that of neural crest cells, which was the origin of the dorsal root ganglia and/or abnormal polarity of the somite, this mouse model with spinal nerve defects at high incidence would be useful to examine the effects of valproic acid on the somitogenesis and morphogenesis of somite-associated structures.

Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies.

Histopathologic and immunohistochemical features of soft palate muscles and nerves in dogs with an elongated soft palate.

To histologically evaluate and compare features of myofibers within the elongated soft palate (ESP) of brachycephalic and mesocephalic dogs with those in the soft palate of healthy dogs and to assess whether denervation or muscular dystrophy is associated with soft palate elongation. Soft palate specimens from 24 dogs with ESPs (obtained during surgical intervention) and from 14 healthy Beagles (control group). All the soft palate specimens underwent histologic examination to assess myofiber atrophy, hypertrophy, hyalinization, and regeneration. The degrees of atrophy and hypertrophy were quantified on the basis of the coefficient of variation and the number of myofibers with hyalinization and regeneration. The specimens also underwent immunohistochemical analysis with anti-neurofilament or anti-dystrophin antibody to confirm the distribution of peripheral nerve branches innervating the palatine myofibers and myofiber dystrophin expression, respectively. Myofiber atrophy, hypertrophy, hyalinization, and regeneration were identified in almost all the ESP specimens. Degrees of atrophy and hypertrophy were significantly greater in the ESP specimens, compared with the control specimens. There were fewer palatine peripheral nerve branches in the ESP specimens than in the control specimens. Almost all the myofibers in the ESP and control specimens were dystrophin positive. These results suggested that palatine myopathy in dogs may be caused, at least in part, by denervation of the palatine muscles and not by Duchenne- or Becker-type muscular dystrophy. These soft palate changes may contribute to upper airway collapse and the progression of brachycephalic airway obstructive syndrome.

Successful survival in culture of spinal cord (SC) dorsal root ganglia (DRG) neurons and glia cells after automatic vitrification

To assess the survival and re-growth of neuronal cells after cryopreservation with a new automatic vitrification system. Experimental research with SC and DRG isolated from rat fetuses. Stationary organotypic DRG and SC cultures were prepared from rat fetuses (gestational day 15, Lewis inbred, Harlan, Israel). mmediately after dissection, the DRG and SC were cut into small slices (400 micron) and seeded in 12 well culture plates containing 1 mL of NVR-Gel. Cultures were enriched once in gel with GDNF conjugated iron oxide nanoparticles (10 ng/mL) and subsequently with nutrient medium at each consecutive feeding. Monitoring of the DRG-SC growth pattern was done by daily phase contrast microscopic observations 24 hours after setting the cultures onward and by IF staining. The various neuronal samples (SC, DRG, glia cells) were cryopreserved with a new device for automatic vitrification (Sarah®, Fertilesafe, IL), consisting of a special capsule containing EM gold grids attached to 0.25ml straws (IMV,France). The straws loaded with neuronal tissue were placed into the mixing chamber of the device attached to a syringe pump dispensing timed and gradual increasing concentrations of equilibrium solution for 12-25 min. and vitrification solutions for 3-5 min., before plunginginto liquid nitrogen slush (VitMaster, IMT IL). After 1 week the samples were rewarmed according to the Origio’s kit instructions. To assess survival, the cells recovered were fixed (4% paraformaldehyde), permeabilized with 0.1% Triton X-100 in PBS and then immunoblocked with a 1% BSA in PBS for 1 h at RT prior to double staining with mouse anti S-100 antibodies (1:80, Acris Antibodies, glial cell marker) and rabbit anti neurofilament antibodies (NF, Novus Biologicals, 1:500, neuronal cell marker). The primary antibodies were diluted in 0.1% BSA and 0.05% Tween-20 in PBS and incubated with the specimens overnight at 4 °C. After rinsing, the DRG specimens were incubated for 1 h at RT with fluorescent secondary antibodies. Organotypic cultures of SC, DRG neurons and glia cells demonstrated a remarkable survival as well as nerve fiber regeneration after cryopreservation/rewarming. The SC neurons maintained their multipolar shape with re-growth of dendrites and axons. The round shaped DRG neurons exhibited euchromatic nuclei with prominent nucleoli and an active regeneration of nerve processes. This is the first report on successful vitrification of SC cells, DRG neurons and glia cells using an automatic freezing device. The remarkable resumption of growth for neuronal cells will greatly expand regenerative research opportunities.

Cholecalciferol (vitamin D3) improves functional recovery when delivered during the acute phase after a spinal cord trauma

In a previous study, based on a rat model of thoracic spinal cord compression, we demonstrated that cholecalciferol (Vitamin D3), delivered at the dose of 200IU/kg/day, significantly improved ventilatory frequency and spasticity. In order to confirm the restorative potential of vitamin D, we performed a new study, using a rat model of left cervical hemisection (C2). From Day 1 or Day 7, animals received, during three months, a weekly oral bolus of either cholecalciferol, at the dose of 500IU/kg/day, or vehicle, namely triglycerides. Rats were assessed every month, using a ladder test for sensori-locomotor ability and neuromuscular capacity. Three months after injury, H-reflex was recorded from left extensor digitorum muscle in order to measure the reflexivity of the sub-lesional region. Ventilatory frequency was also monitored during an electrically induced muscle fatigue of the hindlimb known to enhance muscle metaboreflex and increase respiratory rate. After recording the phrenic nerve activity, ipsilateral to the lesion, during spontaneous breathing, animals were artificially ventilated while paralyzed with a neuromuscular blocking agent and then the brainstem respiratory centres were provoked to maximal output by temporarily stopping the ventilator. Spinal cords were immunostained with an anti-neurofilament antibody to evaluate axon numbers. We show here that vitamin D-treated animals display i) an enhanced locomotor activity, ii) an improved breathing when hindlimb muscle was electrically stimulated to induce fatigue, iii) an H-reflex depression similar to control animals, iv) a phrenic nerve activity response to a temporary asphyxial stress and v) a non significant decreased number of axons in the proximal stump when compared with the Sham group. This new set of data confirms that vitamin D is a potent molecule that could be tested in clinical trials assessing functional recovery in para-/tetra-plegic patients, shortly after a trauma.

Quantitative correlation between cardiac MIBG uptake and remaining axons in the cardiac sympathetic nerve in Lewy body disease

ObjectivesReduced cardiac meta-iodobenzylguanidine (MIBG) uptake and loss of cardiac sympathetic axons, as its possible anatomical substrate, were both recognised in Lewy body disease (LBD), while their direct correlation has so...

Biological aspects of appendix pathology: irregular distribution of myenteric ganglia in human appendiceal wall

Although appendicitis is a common disease, basic questions about risk factors and its etiology remain unexplained. An obstruction of the appendix lumen is usually considered to be the main cause of acute appendicitis. However, more studies are currently dealing with neuroimmune appendicitis. We studied samples of human appendices with the histological diagnosis of chronic appendicitis. Fixed cryosections of appendiceal walls were examined by immunofluorescence methods using neuronal anti-neurofilament antibody markers and beta III tubulin. The immunostaining revealed an irregular distribution of myenteric ganglia in inflamed appendiceal walls and unexpected groups of large ganglia unequally distributed in the subserosal area. The comparative analysis of normal and inflamed appendix samples showed differences in the occurrence of myenteric ganglia in the subserosal area. They appeared more frequently on cryosections prepared from the inflamed appendiceal wall. We propose that the high variability and irregular location of myenteric ganglia in the appendiceal wall are due to an alteration in the motility which results in flaccid appendix emptying. In addition, superficially located myenteric ganglia are exposed to abdominal irritation and may explain the chronic abdominal pain which is often considered to be a sign of chronic appendicitis (Fig. 2, Ref. 23).

Development of a novel method for decellularizing a nerve graft using a hypertonic sodium chloride solution.

Acellular nerves are a reconstruction material and provide scaffolds for nerve regeneration. Numerous methods to develop acellular nerves have been described. However, these methods pose problems that can be attributed to incomplete acellular processing and destruction of the extracellular matrix (ECM); the former may lead to rejection response, while the latter may damage the scaffold. In order to overcome problems associated with the above-mentioned methods, we developed a novel method that employs a hypertonic sodium chloride solution to decellularize nerve graft material. Rat sciatic nerves were harvested, dipped in hypertonic sodium chloride solution (1 M), and shaken for 24 h. We then washed the nerves in phosphate-buffered saline for 7 days with shaking and evaluated the acellular nerves by hematoxylin-eosin (H-E) staining, immunostaining, and electron microscopy. We then transplanted the grafts to the sciatic nerve of another rat and evaluated the outcomes by H-E staining, immunostaining (anti-neurofilament antibody, anti-S-100 antibody), anterograde nerve tracing, and electron microscopy. We found that our method successfully decellularized the grafts, but was mild enough to leave the ECM intact. Two months after transplantation, immunostaining and anterograde nerve tracing confirmed that Schwann cells infiltrated the grafts and induced neurofilament extension. Our methodology preserves the ECM, is simple to develop, and does not involve substances that harm biogenic tissue. Acellular nerve tissue processed in this way could become a substitute material for bridging nerve gaps. Our method could also aid in the development of other acellular tissues.

Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.

Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.

Neurogenesis in the lamprey central nervous system following spinal cord transection

After spinal cord transection, lampreys recover functionally and axons regenerate. It is not known whether this is accompanied by neurogenesis. Previous studies suggested a baseline level of nonneuronal cell proliferation in the spinal cord and rhombencephalon (where most supraspinal projecting neurons are located). To determine whether cell proliferation increases after injury and whether this includes neurogenesis, larval lampreys were spinally transected and injected with 5-bromo-2&prime-deoxyuridine (BrdU) at 0-3 weeks posttransection. Labeled cells were counted in the lesion site, within 0.5 mm rostral and caudal to the lesion, and in the rhombencephalon. One group of animals was processed in the winter and a second group was processed in the summer. The number of labeled cells was greater in winter than in summer. The lesion site had the most BrdU labeling at all times, correlating with an increase in the number of cells. In the adjacent spinal cord, the percentage of BrdU labeling was higher in the ependymal than in nonependymal regions. This was also true in the rhombencephalon but only in summer. In winter, BrdU labeling was seen primarily in the subventricular and peripheral zones. Some BrdU-labeled cells were also double labeled by antibodies to glial-specific (antikeratin) as well as neuron-specific (anti-Hu) antigens, indicating that both gliogenesis and neurogenesis occurred after spinal cord transection. However, the new neurons were restricted to the ependymal zone, were never labeled by antineurofilament antibodies, and never migrated away from the ependyma even at 5 weeks after BrdU injection. They would appear to be cerebrospinal fluid-contacting neurons.