Neurofilament Protein Immunostaining Research Articles

Histological identification of nasopharyngeal mechanoreceptors.

The auditory tube plays a fundamental role in regulating middle ear pressure. A "system" sensitive to a pressure gradient between the middle ear and the ambient environment is necessary. The presence of mechanoreceptors in the middle ear and the tympanic membrane has been studied, but the presence of these receptors in the nasopharyngeal region remains unclear. The aim of this study is to confirm the presence of pressure sensitive corpuscles in the nasopharynx. An experimental study was conducted on five fresh and unembalded human cadavers. The pharyngeal ostium of the auditory tube and its periphery was removed in one piece by video-assisted endonasal endoscopy. Samples were fixed in formaldehyde solution, embedded in paraffin, and cut. Slides were analyzed by HES (Hematoxyline Eosine Safran) coloration, by S100 protein and neurofilament protein immunostaining. Encapsulated nerve endings were researched and identified by slides analysis. Eight samples were included in our study. On seven samples, Ruffini corpuscles were identified in the mucosa of the posterior area of the pharyngeal ostium, with a higher concentration in the pharyngeal recess and in the posterior nasopharyngeal wall. Our study identified nasopharyngeal mechanoreceptors that could detect the nasopharyngeal pressure and, by extension, the atmospheric pressure. These findings support the theory of the neuronal reflex arc of isobaric system of the middle ear, based on the existence of a "system" sensitive to a pressure gradient between the middle ear and the ambient environment. Understanding of this system has been helpful in the diagnosis and management of middle ear diseases.

Morphological assessment of renal arteries after radiofrequency catheter-based sympathetic denervation in a porcine model

Catheter-based renal artery denervation has been successfully introduced as alternative treatment for patients suffering from drug-resistant essential hypertension. However, the local morphological changes within the vessel wall accompanying this technique remain elusive and we sought to characterize these by utilizing the simplicity radiofrequency catheter approach. Following treatment of seven pigs, renal arteries were assigned to either the acute (n = 6), subacute (10-day follow-up, n = 6) or control (untreated, n = 2) group. At follow-up blood analysis, final angiography and optical coherence tomography (OCT)-imaging of the treated arteries were performed and renal arteries and kidneys were processed for histopathology and immunohistochemistry. Radiofrequency-derived energy application to the vessel wall induced transmural tissue coagulation and loss of endothelium resulting in local thrombus formation also detectable by OCT. At 10 days, the luminal surface was almost completely re-endothelialized. Mural wall damage was replaced by fibrotic tissue and the adventitial layer showed strong inflammatory infiltration including vasculogenesis. Remnant autonomic nerve fascicles within the lesion segments of the subacute group displayed enhanced vacuolic degeneration and an impaired neurofilament protein immunostaining pattern. Examination of the kidneys revealed no abnormalities and blood parameters remained within the physiological range. Catheter-based application of radiofrequency energy resulted in circumscribed transmural injury within the arterial wall affecting autonomic nerve fascicles delayed to treatment. Acute loss of endothelialization resulted in thrombus formation leaving kidney perfusion apparently unimpaired.

Brain stem gliomas: a clinicopathological study from a single cancer center

Brain stem gliomas (BSG) are rare tumors occurring predominantly in childhood. They are mostly of astrocytic origin and are divided into infiltrative versus circumscribed types, with different prognoses. The diagnosis is mainly based on MRI findings, and biopsy is rarely performed. This is a retrospective study of BSG with available biopsies diagnosed at our center over 6-year period. Fifteen cases were identified, with a predominance of females. The median age was 7 years, and the mean duration of symptoms was <6 weeks in 58.3% (n = 7) of cases. MRI was typical of diffuse pontine gliomas in 64.3% (n = 9) of cases. Radiotherapy was the commonest modality of treatment, and the median overall survival was 21.7 months. Twelve cases were consistent with infiltrative astrocytoma of various grades (2 grade II, 7 grade III and 3 grade IV). Entrapped normal neurons and mitosis were the commonest findings indicating infiltrative growth and high grade, respectively, and those correlated significantly with immunostaining for neurofilament protein and Ki-67 of ≥3%. Overall survival correlated only with the duration of symptoms and tumor grade on biopsies. A limited panel of immunostains might be useful in undetermined cases to decide on the growth pattern and grade.

Can Dynamic Contrast-Enhanced Magnetic Resonance Imaging Combined with Texture Analysis Differentiate Malignant Glioneuronal Tumors from Other Glioblastoma?

An interesting approach has been proposed to differentiate malignant glioneuronal tumors (MGNTs) as a subclass of the WHO grade III and IV malignant gliomas. MGNT histologically resemble any WHO grade III or IV glioma but have a different biological behavior, presenting a survival twice longer as WHO glioblastomas and a lower occurrence of metastases. However, neurofilament protein immunostaining was required for identification of MGNT. Using two complementary methods, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and texture analysis (MRI-TA) from the same acquisition process, the challenge is to in vivo identify MGNT and demonstrate that MRI postprocessing could contribute to a better typing and grading of glioblastoma. Results are obtained on a preliminary group of 19 patients a posteriori selected for a blind investigation of DCE T1-weighted and TA at 1.5 T. The optimal classification (0/11 misclassified MGNT) is obtained by combining the two methods, DCE-MRI and MRI-TA.

Routine Use of Immunohisto-chemistry May Increase the Frequency of Hybrid Peripheral Nerve Sheath Tumors

Routine Use of Immunohisto-chemistry May Increase the Frequency of Hybrid Peripheral Nerve Sheath Tumors

Intracerebral schwannoma in a child with infiltration along perivascular spaces resembling meningioangiomatosis

Schwannoma arising within brain parenchyma is a rare lesion, usually found in children. Reported herein is a case of intracerebral schwannoma in a 5-year-old boy, with a review of the English-language literature on the subject, in which 47 cases were found. Few detailed histological reviews of intracerebral schwannoma exist. The tumor had a distinctive plexiform growth pattern, and small aggregates of Schwann cells spread extensively into the surrounding brain tissue along perivascular spaces adjacent to the tumor nodule. Histological differential diagnoses included perivascular schwannosis and meningioangiomatosis. A few intratumoral axons, seen on immunostaining for neurofilament protein, were trapped at the periphery of the main lesion, but there was no evidence of intralesional axons in the multiple nodules of Schwann cell proliferations that extended into the perivascular spaces, suggesting that the lesions are neoplastic. Because Schwann cells are not a natural component of the central nervous system, the origin of intracerebral schwannomas remains unknown. The histology suggests that Schwann cells of the perivascular nerve plexus are a likely site of origin.

A growth hormone-secreting adenoma with incomplete nerve bundle formation

We present a unique case of an adenoma secreting growth hormone (GH), showing incomplete nerve bundle formation without ganglion cells. A 47-year-old man presenting with acromegaly was revealed to have high serum GH and IGF-1 levels. The concentrations of the other adenohypophysial hormones were within the normal range. Histology revealed an unusual pituitary adenoma containing many nerve bundle-like structures. Adenoma cells with ovoid or round hyperchromatic nuclei and eosinophilic cytoplasms lacked the typical features of ganglion cells. The nerve bundles consisted of slender elongated cells. These fibers were arranged into groups in a roughly parallel fashion. By immunohistochemistry, many adenoma cells were positive for GH, prolactin, thyrotropin beta, synaptophysin and chromogranin. Fibrous bodies revealed by keratin immunostaining were found only in adenoma cells. Scattered star-shaped adenoma cells showed the same immunoreactivity as folliculo-satellite cells. Adenoma cells, but not the bundle-like structures, were also positive for Pit-1. Immunostaining for neurofilament protein, GFAP, vimentin, and S-100 protein revealed variable amounts of fibrils within the bundle-like structures. Scattered immunoreactivity for myelin basic protein and synaptophysin was also found in the bundle area. Our case is the first GH-secreting pituitary adenoma showing incomplete nerve bundle differentiation and lacking mature ganglion cells.

The Controversial Nosology of Benign Nerve Sheath Tumors: Neurofilament Protein Staining Demonstrates Intratumoral Axons in Many Sporadic Schwannomas

Schwannomas are benign peripheral nerve sheath tumors believed to be composed purely of cells with ultrastructural features of Schwann cells; these tumors are believed to develop eccentrically from the surface of nerves and not to contain axons, other than immediately beneath the capsule. This concept has recently been disputed in cases associated with neurofibromatosis type 2. The usual presence of intratumoral axons in neurofibromas is said to allow easy distinction from schwannomas. Eighty sporadic schwannomas (20 conventional, 20 cellular, 20 ancient, 10 gastric, and 10 plexiform) were retrieved from the authors' files. Hematoxylin-and-eosin stained slides were reviewed, diagnoses were confirmed and all tumors were stained for S-100 protein and neurofilament protein (NFP). The amount (rare, focal, multifocal, and diffuse) and distribution (central and/or peripheral) of axons within the tumors were analyzed. All tumors were strongly and diffusely positive for S-100 protein (nuclear and cytoplasmic staining). NFP-positive axons were identified in 11 of 20 (55%) conventional schwannomas (2 rare, 4 focal, 3 multifocal, and 2 diffuse; 5 central, 4 peripheral, and 2 central and peripheral) and in 15 of 20 (75%) cellular schwannomas (3 rare, 6 focal, and 6 multifocal; 12 central, 1 peripheral, and 2 central and peripheral). Of the 20 ancient schwannomas, 7 cases (35%) showed intratumoral axons, highlighted by NFP immunostaining (1 rare, 4 focal, 1 multifocal, and 1 diffuse; 4 peripheral, 2 central, and 1 central and peripheral). Most cases of gastric schwannoma showed no evidence of intratumoral axons; 9 cases (90%) were negative for NFP and only 1 case (10%) was positive (focal and central). Seven of 10 cases (70%) of plexiform schwannomas were negative for NFP, whereas only 3 cases (30%) showed positive axons (2 multifocal and 1 focal; 3 central). The unexpected but quite frequent presence of intratumoral axons in schwannomas argues against conventional views of these lesions' pathogenesis as an eccentric encapsulated lesion and raises the possibility that a more diverse cell population, perhaps more closely resembling neurofibromas, may constitute these neoplasms. Although NFP-positive axons were most often present in the conventional and cellular variants of schwannoma, their presence was also observed in a minority of ancient, gastric and plexiform schwannomas. Differentiation between neurofibroma and schwannoma in cases with overlapping cytoarchitectural features should not be based solely on the presence or absence of NFP-positive axons within a given tumor.

Decreased neural damage after spinal cord injury in tPA-deficient mice.

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin. It plays an important role in the nervous system, including the processes of neuronal migration, neurite outgrowth, and neuronal plasticity. tPA has also been suggested to have a role in several neuropathological conditions, such as cerebral ischemia, seizures, and demyelinating diseases. To investigate the role of tPA in spinal cord injury, wild-type mice and mice with homozygous tPA deficiency (tPA(-/-) mice) were subjected to spinal cord contusion and the differences of hindlimb function, electrophysiological changes, and histopathological changes were assessed for 6 weeks. Functional recovery was greater in tPA(-/-) mice than in wild-type mice throughout the observation period. The time course of myoelectric motor-evoked potentials supported the hindlimb functional findings. Histological examination showed that injured areas were smaller in tPA(-/-) mice than wild-type mice on Luxol fast blue staining or myelin basic protein and neurofilament protein immunostaining at 6 weeks after contusion. Electron microscopy showed that the white matter was better preserved in tPA(-/-) mice than in wild-type mice. The expression of tPA protein was widespread on the first day after contusion and this expression was detected for at least a week. Activation of microglia/macrophages and apoptotic cell death were significantly reduced in tPA(-/-) mice after contusion. This study shows that neural damage is decreased in tPA(-/-) mice after spinal cord injury. Suppression of tPA production may help to decrease secondary injury after spinal cord contusion.

Reactive glia support and guide axon growth in the rat thalamus during the first postnatal week. A sharply timed transition from permissive to non-permissive stage

The present study demonstrates a supportive and guiding effect of the reactive glia on the postlesional axon growth in vivo, and offers a model system to compare permissive and non-permissive forms of the glial reaction. After stab wounds in early postnatal (P2–P9) rats, the reactive glia and the nerve fibers were detected by the immunohistochemical staining of glial fibrillary acidic protein (GFAP) and neurofilament protein, respectively. In the thalamus of the animals lesioned at P5 or earlier, an extraordinary bundle of fibers immunoreactive to neurofilament protein was found, corresponding to the lesion track marked by reactive glia. This bundle persisted up to 2 months, as shown by electron microscopy. When the animals were lesioned at P7 or later, the lesion track was immunonegative to neurofilament protein. Following P6 lesions, an intermediate situation was found, the strip of immunoreactive neurofilament protein was missing, or short and weak. GFAP immunostaining demonstrated a typical reactive glia in every case. As a result of the same operation, reactive glia plus a deficiency of neurofilament protein immunostaining was found in every animal in the cortex and the corpus callosum, independently from the age at lesion. The results demonstrate that the permissive nature of the glial reaction depends on the lesioned area as well, and changes to a non-permissive effect in a short time interval.

Olfaction preservation in anterior cranial base approaches: an anatomic study.

To study the anatomic basis for olfaction-sparing anterior cranial base approaches. The medial anterior skull base containing the olfactory unit and delimited by the inner table of the frontal sinus, the lesser wing of the sphenoid bone, and the medial orbital walls was removed from six cadaveric specimens. Histological methods were used to investigate the location, distribution, and depth of penetration of olfactory nerves. Hematoxylin and eosin and Gomori trichrome staining were used to visualize landmarks and architecture. S-100 neurofilament protein immunostaining was used to identify nerve fascicles and axons. In three cadaveric head specimens, olfaction-sparing craniofacial approaches were performed and the excised olfactory units were evaluated histologically. Bundles of olfactory nerves were identified primarily in the nasal septum; relatively fewer bundles could be identified in the middle turbinate. Olfactory nerve endings were identified up to 20 mm below the cribriform plate (range, 7-20 mm). The superior and middle nasal meatus were most innervated; olfactory innervation was virtually absent in the inferior nasal meatus. Histological evaluation of the olfactory unit elevated during olfaction-sparing techniques routinely revealed transection of olfactory nerves that exited the skull base. In olfaction-sparing anterior cranial base approaches, the olfactory nerves are inevitably transected. The clinical significance of olfactory nerve transection for postoperative functional recovery of olfaction remains to be analyzed.

Perikaryal accumulation and proteolysis of neurofilament proteins in the post-mortem rat brain

Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved α-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition.

Neurofilament reorganisation and neurofilament antigen redistribution in spinal motoneurones following retrograde axonal transport of diphtheria toxin.

Single unilateral injections of diphtheria toxin (DTX) into the external anal sphincter muscle or internal intercostal nerve of cat induced characteristic ultrastructural lesions in corresponding ipsilateral spinal motoneurones 6-8 days later. The chief neuronal lesion was a progressive disruption of Nissl body composition and organisation, which between days 8-19 post injection was accompanied by a progressive accumulation of neurofilaments in motoneuronal perikarya and dendrites. Some axons in the ipsilateral ventral horn became hypertrophied due to neurofilamentous accumulation. Related immunocytochemical investigations 6-35 days after injection of DTX revealed abnormal immunoreactivity intoxicated motoneurones for 200-kDa and 160-kDa phosphorylated neurofilament proteins, but not in contralateral motoneurones. By day 35 abnormal neurofilament immunostaining also occurred in ipsilateral and some contralateral interneurones but not contralateral motoneurones. Abnormalities of Nissl body endoplasmic reticulum, neurofilament organisation, and neurofilament protein immunostaining were identical after either intraneural and intramuscular injections of DTX, indicating abnormalities were attributable to toxicity and not injection-related axonal damage. Since DTX acts specifically in the soma to inhibit protein synthesis, neurofilament abnormalities are secondary to cytotoxicity and probably result from deficits in transference of existing partially phosphorylated neurofilaments to the axonal transport system, or axonal transport per se.

Acrylamide-induced visceral neuropathy: Evidence for the involvement of capsaicin-sensitive nerves of the rat urinary bladder

The mechanisms underlying the severe urinary retention induced by acrylamide intoxication were studied in detail in the rat. Subcutaneous treatment with acrylamide monomer (50 mg/kg daily for 10 days) almost completely impaired the micturition reflex, resulting in urinary retention. In fact, the ability to eliminate an oral water load was virtually abolished, while bladder filling with saline (transvesical cystometrogram) failed to activate reflex micturition. Instead, a picture of overflow incontinence resulted in urethane-anaesthetized rats, which was not reversed by intravenous administration of 4-aminopyridine. The nerve-mediated contractile response to field stimulation (0.1–20 Hz, 0.5ms, 60V) of the isolated bladder was unaffected, thus suggesting the integrity of bladder efferent innervation, and no evidence was found from in vitro experiments that the myogenic contractility of the bladder was depressed by acrylamide treatment. Conversely, the sensory nerve-mediated response to capsaicin was abolished and sensory nerve fibres of the bladder were selectively depleted of their content of substance P- and calcitonin gene-related peptide immunoreactivity following acrylamide treatment. In fact, concentrations of the same neuropeptides in other organs, including the adjoining ureters, were unaffected. As to the urethral segment, including the striated sphincter, the d-tubocurarine (0.2 mM)-sensitive urethral response to electrical stimulation (0.1 Hz, 0.1 ms, 20 V) was significantly reduced in acrylamide-treated animals. At the same level, neurofilament protein immunostaining revealed striking accumulations of neurofilament protein-like material in motor end-plates, thus indicating that neuromuscular junctions of the urethral striated sphincter were severely affected. Thus, the afferent arm of the micturition reflex was shown to be severely deranged by acrylamide intoxication, especially in its capsaicin-sensitive component. Since twitch-like contractions of the urethral striated sphincter are probably involved in promoting bladder voiding, a decreased efficiency of this mechanism could participate in the picture of urinary retention induced by acrylamide.

Responses of pulpal nerves to cavity preparation in rat molars: An immunohistochemical study using neurofilament protein(NFP) antiserum.

The response of neural elements to a dentin injury was morphologically investigated in rat molars by use of immunostaining for neurofilament protein (NFP). An artificially formed cavity in dentin by drilling rapidly caused the displacement of some odontoblasts into the exposed dentinal tubules, while others were detached from the predentin. The subodontoblastic nerve plexus consisting of NFP-immunoreactive nerves shifted inward together with the separated odontoblasts, while a movement of the nerves into the exposed dentinal tubules was not recognized. The odontoblasts separated from the predentin degenerated and disappeared one day after the cavity preparation; at this time, the subodontoblastic nerve plexus underlying the drilled dentin was remarkably disrupted, presumably losing dentinal sensation of the drilled area. Three days after the cavity preparation, the destroyed odontoblastic layer began to be repaired by newly differentiating odontoblasts; the reparative dentin was produced from 5 to 7 days onward. Numerous NFP-positive nerves, beaded in type, gathered in the odontoblastic layer in accordance with the differentiation of the new odontoblasts. The increased beaded nerve fibers were suggested to represent peptide-containing nerves. In 10-15 days, the reparative dentin accumulated quite remarkably under the cavity area. The NFP-positive subodontoblastic nerve plexus was entirely reconstituted and also regained continuity to its surrounding plexus. The nerve fibers in the reconstituted plexus were mostly non-beaded in type as seen in the control teeth. Since dentinal tubules in the reparative dentin are not normally continuous to the primary dentinal tubules, dentinal sensation may not have been restored.

Sensory innervation of the periodontal ligament in the incisor and molar of the monkey, Macaca fuscata. An immunohistochemical study for neurofilament protein and glia-specific S-100 protein.

Nervous elements in the periodontal ligament of the monkey, Macaca fuscata were investigated by means of immunohistochemistry for neurofilament protein (NFP) and S-100 protein. Thick nerve bundles with NFP-immunoreactivity entered the periodontal ligament through slits at the bottom of the alveolar socket, whereas thinner bundles, also NFP-immunoreactive, penetrated the ligament from the lateral wall of the alveolar socket. The NFP-immunopositive nerve fibers were densely distributed around the root apex in both incisors and molars. In the upper incisors, another dense distribution of NFP-positive nerves was found in the apical half of the lingual periodontal ligament and in the coronal half of the labial periodontal ligament. The periodontal nerve fibers in the incisors ramified in a dendritic fashion, terminating as free nerve endings within the periodontal ligament. Immunostaining for S-100 protein demonstrated the Schwann sheaths of nerves which showed the same distribution pattern as that of the NFP-immunopositive fibers in the periodontal ligament of the incisors. In molars, immunostaining for NFP and S-100 showed that nerves were numerous in the apical region of the periodontal ligament. However, NFP-immunopositive nerves were fewer than the S-100-positive nervous elements in the apical region in molars. This discrepancy suggests the presence of NFP-immunonegative nerves. Immunostaining for S-100 is thus useful for demonstration of neural elements in the periodontal ligament of molars. The S-100-immunoreactive neural elements in the periodontal ligament were more numerous in incisors than in molars. In addition to the free nerve endings, a small population of coiled nerve endings was observed in the periodontal ligament of molars. Most of the nerve fibers and cell bodies in the trigeminal ganglion showed NFP-immunoreactivity, whereas all in the mesencephalic nucleus of the trigeminal nerve were negative in NFP-reaction. This finding suggests that the periodontal ligament of molars receives a dual innervation from the trigeminal ganglion and mesencephalic nucleus.