Axonal Degeneration in Dental Pulp Precedes Human Primary Teeth Exfoliation.

The dental pulp in human primary teeth is densely innervated by a plethora of nerve endings at the coronal pulp-dentin interface. This study analyzed how the physiological root resorption (PRR) process affects dental pulp innervation before exfoliation of primary teeth. Forty-four primary canine teeth, classified into 3 defined PRR stages (early, middle, and advanced) were fixed and demineralized. Longitudinal cryosections of each tooth were stained for immunohistochemical and quantitative analysis of dental pulp nerve fibers and associated components with confocal and electron microscopy. During PRR, axonal degeneration was prominent and progressive in a Wallerian-like scheme, comprising nerve fiber bundles and nerve endings within the coronal and root pulp. Neurofilament fragmentation increased significantly during PRR progression and was accompanied by myelin degradation and a progressive loss of myelinated axons. Myelin sheath degradation involved activation of autophagic activity by Schwann cells to remove myelin debris. These cells expressed a sequence of responses comprising dedifferentiation, proliferative activity, GAP-43 overexpression, and Büngner band formation. During the advanced PRR stage, increased immune cell recruitment within the dental pulp and major histocompatibility complex (MHC) class II upregulation by Schwann cells characterized an inflammatory condition associated with the denervation process in preexfoliative primary teeth. The ensuing loss of dental pulp axons is likely to be responsible for the progressive reduction of sensory function of the dental pulp during preexfoliative stages.

Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries.

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment.

Antiglaucomatous effects of the activation of intrinsic Angiotensin-converting enzyme 2.

PURPOSE: To evaluate the effects of the activation of endogenous angiotensin-converting enzyme 2 (ACE2) using the compound diminazene aceturate (DIZE) in an experimental model of glaucoma in Wistar rats. METHODS: DIZE (1 mg/kg) was administered daily, either systemically or topically, and the IOP was measured weekly. To examine the role of the Mas receptor in the effects of DIZE, the Ang-(1-7) antagonist A-779 was co-administered. Drainage of the aqueous humor was evaluated by using scintigraphy. The analysis of ACE2 expression by immunohistochemistry and the counting of retinal ganglion cells (RGCs) were performed in histologic sections. Additionally, the nerve fiber structure was evaluated by transmission electron microscopy. RESULTS: The systemic administration and topical administration (in the form of eye drops) of DIZE increased the ACE2 expression in the eyes and significantly decreased the IOP of glaucomatous rats without changing the blood pressure. Importantly, this IOP-lowering action of DIZE was similar to the effects of dorzolamide. The antiglaucomatous effects of DIZE were blocked by A-779. Histologic analysis revealed that the reduction in the number of RGCs and the increase in the expression of caspase-3 in the RGC layer in glaucomatous animals were prevented by DIZE. This compound also prevented alterations in the cytoplasm of axons in glaucomatous rats. In addition to these neuroprotective effects, DIZE facilitated the drainage of the aqueous humor. CONCLUSIONS: Our results evidence the pathophysiologic relevance of the ocular ACE2/Ang-(1-7)/Mas axis of the renin-angiotensin system and, importantly, indicate that the activation of intrinsic ACE2 is a potential therapeutic strategy to treat glaucoma.

Anatomy of corpus callosum in prenatally malnourished rats.

The effect of prenatal malnutrition on the anatomy of the corpus callosum was assessed in adult rats (45-52 days old). In the prenatally malnourished animals we observed a significant reduction of the corpus callosum total area, partial areas, and perimeter, as compared with normal animals. In addition, the splenium of corpus callosum (posterior fifth) showed a significant decrease of fiber diameters in the myelinated fibers without changing density. There was also a significant decrease in diameter and a significant increase in density of unmyelinated fibers. Measurements of perimeter's fractal dimensions from sagittal sections of the brain and corpus callosum did not show significant differences between malnourished and control animals. These findings indicate that cortico-cortical connections are vulnerable to the prenatal malnutrition, and suggest this may affect interhemispheric conduction velocity, particularly in visual connections (splenium).

Anatomy of corpus callosum in prenatally malnourished rats

The effect of prenatal malnutrition on the anatomy of the corpus callosum was assessed in adult rats (45-52 days old). In the prenatally malnourished animals we observed a significant reduction of the corpus callosum total area, partial areas, and perimeter, as compared with normal animals. In addition, the splenium of corpus callosum (posterior fifth) showed a significant decrease of fiber diameters in the myelinated fibers without changing density. There was also a significant decrease in diameter and a significant increase in density of unmyelinated fibers. Measurements of perimeter's fractal dimensions from sagittal sections of the brain and corpus callosum did not show significant differences between malnourished and control animals. These findings indicate that cortico-cortical connections are vulnerable to the prenatal malnutrition, and suggest this may affect interhemispheric conduction velocity, particulary in visual connections (splenium).

Microfasciculation: a morphological pattern in leprosy nerve damage.

AIMS: To study Microfasciculation, a perineurial response found in neuropathies, emphasizing its frequency, detailed morphological characteristics and biological significance in pure neural leprosy (PNL), post-treatment leprosy neuropathy (PTLN) and non-leprosy neuropathies (NLN). METHODS AND RESULTS: Morphological characteristics of microfascicles were examined via histological staining methods, immunohistochemical expression of neural markers and transmission electronmicroscopy. The detection of microfasciculation in 18 nerve biopsy specimens [12 PNL, six PTLN but not in the NLN group, was associated strongly with perineurial damage and the presence of a multibacillary inflammatory process in the nerves, particularly in the perineurium. Immunoreactivity to anti-S100 protein, anti-neurofilament, anti-nerve growth receptor and anti-myelin basic protein immunoreactivity was found within microfascicles. Ultrastructural examination of three biopsies showed that fibroblast-perineurial cells were devoid of basement membrane despite perineurial-like NGFr immunoreactivity. Morphological evidence demonstrated that multipotent pericytes from inflammation-activated microvessels could be the origin of fibroblast-perineurial cells. CONCLUSIONS: A microfasciculation pattern was found in 10% of leprosy-affected nerves. The microfascicles were composed predominantly of unmyelinated fibres and denervated Schwann cells (SCs) surrounded by fibroblast-perineurial cells. This pattern was found more frequently in leprosy nerves with acid-fast bacilli (AFB) and perineurial damage while undergoing an inflammatory process. Further experimental studies are necessary to elucidate microfascicle formation.

Forebrain projections to brainstem nuclei involved in the control of mandibular movements in rats.

Mandibular movements occur through the triggering of trigeminal motoneurons. Aberrant movements by orofacial muscles are characteristic of orofacial motor disorders, such as nocturnal bruxism (clenching or grinding of the dentition during sleep). Previous studies have suggested that autonomic changes occur during bruxism episodes. Although it is known that emotional responses increase jaw movement, the brain pathways linking forebrain limbic nuclei and the trigeminal motor nucleus remain unclear. Here we show that neurons in the lateral hypothalamic area, in the central nucleus of the amygdala, and in the parasubthalamic nucleus, project to the trigeminal motor nucleus or to reticular regions around the motor nucleus (Regio h) and in the mesencephalic trigeminal nucleus. We observed orexin co-expression in neurons projecting from the lateral hypothalamic area to the trigeminal motor nucleus. In the central nucleus of the amygdala, neurons projecting to the trigeminal motor nucleus are innervated by corticotrophin-releasing factor immunoreactive fibers. We also observed that the mesencephalic trigeminal nucleus receives dense innervation from orexin and corticotrophin-releasing factor immunoreactive fibers. Therefore, forebrain nuclei related to autonomic control and stress responses might influence the activity of trigeminal motor neurons and consequently play a role in the physiopathology of nocturnal bruxism.

New insights on the olfactory lobe of decapod crustaceans.

Studies on the crustacean nervous system have been responsible for advances in neuroscience and continue to play an important role in comparative neurobiology. The organization of crustacean brains provides clues about their phylogeny and might also offer some hints regarding their lifestyles. In this study, we compared the organization of specific brain regions of three species of marine shrimp with two species of freshwater prawns, making possible the identification of a pathway issuing from the periphery and cluster 9, which was not previously identified in freshwater prawns and shrimp. Although the brains of the species studied show the same general organization of other crustaceans, variations in the structural organization of the olfactory lobe, evidenced by different immuno-markers, were observed between shrimp species and prawns. In contrast to shrimp, the more well-defined organization of the olfactory lobe in freshwater prawns might reflect greater integration in the processing of olfactory information. Also, in freshwater prawns we described fibers organized as a tract similar to the deutocerebral commissure.

Bone marrow cells are able to increase vessels number during repair of sciatic nerve lesion.

The aim of this study was to compare the outcomes of nerve autografts (GRF) and venous grafts containing mononuclear bone marrow cells (BMCs) in sciatic nerve-lesioned rats. Control animals underwent sham operations (SHAM), received empty venous grafts (EPV), or received venous grafts containing BMC vehicle (AGR). Outcome was evaluated through sciatic functional index (SFI), morphometric and morphologic analyses of the nerve distal to the lesion, and the number of spinal cord motor neurons positive for the retrograde tracer, Fluoro-Gold. All groups exhibited poor results in SFI when compared to SHAM animals throughout the postoperative period. All groups also had a significantly greater fiber density, decreased fiber diameter, and decreased motor neuron number than the SHAM group. No significant difference between the GRF and BMC groups was observed in any of these parameters. On the other hand, vessel density was significantly higher in BMC than all other groups. BMC-containing venous grafts are superior to nerve autografts in increasing vessel density during sciatic nerve regeneration.

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve.

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.

Retinal nerve fibre layer thickness profile in normal eyes using third-generation optical coherence tomography.

AIMS: To establish four normal retinal nerve fibre layer (RNFL) thickness radial profiles based on third-generation optical coherence tomography (OCT) and to compare them with previously reported histologic measurements. METHODS: A total of 20 normal eyes were studied. A circular scan was adjusted to the size of the optic disc and three scans were performed with this radius and every 200 microm thereafter, up to a distance of 1400 microm. Four different radial sections (superotemporal, superonasal, inferonasal, and inferotemporal) were studied to establish RNFL thickness OCT profiles. Additionally, two radial scans orientated at 45 and 135 degrees crossing the optic disc centre were performed in six of 20 eyes, and RNFL thickness was measured at disc margin. RESULTS: Quadrant location and distance from disc margin interaction in RNFL thickness was statistically significant (P<0.001). The RNFL thickness decreased (P<0.001) as the distance from the disc margin increased for all sections. The measurements automatically generated by the OCT built-in software were thinner (P<0.001) than histologic ones close to the disc margin. CONCLUSIONS: Four normal OCT RNFL profiles were established and compared with histological data obtained from the same area. RNFL measurements assessed by OCT 3 were significantly thinner close to the optic disc margin.

Perinatal exposure to androgen suppresses sexual dimorphism in nerve trunk diameter, axon number, and fiber size spectrum: a quantitative ultrastructural study of the adult rat mammary nerve.

The adult mammary nerves (MNs) from female, male, and testosterone-androgenized female rats were studied by light and electron microscopy. The female MNs trunk has twice the diameter of that of the male. Morphometry showed a significantly more myelinated (307 +/- 6) and unmyelinated axons (1654 +/- 10) in the female MN than the male MN (278 +/- 6 and 1373 +/- 28, respectively). Perinatal exposure of the female to testosterone significantly reduced the number of both axon types in the MN in adulthood (244 +/- 6 myelinated and 1300 +/- 32, unmyelinated). Another sexual dimorphism is a distinct group of large (>7.0 microm in diameter) myelinated axons known to conduct sensory information (i.e., touch and vibration). Because the male and the perinatally-androgenized female MNs lack these fibers, it is concluded that gonadal sex hormones may promote the differentiation of specific sets of axons committed to transmission of sensory cues relevant to reproduction.

Ultrastructural study of normal and degenerating nerve fibers in the protocerebral tract of the crab Ucides cordatus.

Wallerian degeneration is a very well described phenomenon in the vertebrate nervous system. In arthropods, and especially in crustaceans, nerve fiber degeneration has not been described extensively. In addition, literature shows that the events do not follow the same patterns as in vertebrates. In this study we report, by qualitative and quantitative ultrastructural analyses, the features and time course of the protocerebral tract degeneration following extirpation of the optic stalk. No remarkable changes were observed seven days after lesion. After 28 days the protocerebral tracts presented apparently preserved small and large diameter axons and some degenerating medium axons, with irregular contours and empty-looking aspect of the axoplasm. Forty days after the ablation of the optic stalks, both small (type I) and medium (type II and III) axons revealed signs of partial or total degeneration, but large nerve fibers (type IV) were still intact. After 45 days, the tract showed signs of advanced stage of degeneration and, apart from large axons, normal-looking fibers were almost absent. At these 3 last time points, degenerating axons displayed different electron densities and aspects, probably correlating to different onset times of the process. In addition, cells with granules in their cytoplasm, possibly hemocytes, were quite distinct, especially at 40 and 45 days after axotomy. These cells might share with glial cells the function of phagocytosis of cellular debris during the protocerebral tract degeneration. Quantitative analysis showed that the number of degenerating fibers increased significantly from 28 to 40 days after lesion, whereas the number of normal fibers decreased accordingly. Measurements of cross-sectional areas of normal and degenerating axons showed that types II and III (medium) start to degenerate before type I (small). Type IV (large) axons do not degenerate, even after 40 days. Therefore, we can conclude that degeneration in these afferent fibers starts late after axotomy, but proceeds at a faster rate afterwards until the complete degeneration of small and medium axons.

Microscopic anatomy of the sural nerve in the postnatal developing rat: a longitudinal and lateral symmetry study.

Rat sural nerve is widely used in experimental studies investigating injury and regeneration of the peripheral nervous system. However, it has not yet been established whether morphological and morphometric parameters differ within corresponding levels of the rat sural nerve. The aims of the present study were to investigate the normal morphological and morphometric aspects of the sural nerve in postnatal developing female rats, with special attention to longitudinal morphology and lateral symmetry. Rats aged 30, 90 and 180 days were killed, and proximal and distal segments of the right and left sural nerves were prepared for light microscopy and morphometric study. No differences were found between the proximal and distal segments or between the right and left sides at the same levels. In addition, postnatal growth continuously and symmetrically affected the sural nerve fascicles and myelinated fibres. Fibre population distribution was also affected by increasing body weight; distribution was unimodal at 30 days, and by 180 days this distribution was established as bimodal. We concluded that the sural nerve is long and constant in its morphology and presents a continuous and symmetrical growth, more pronounced between 30 and 90 days of age, thus providing a good model for experimental neuropathies.

Peripheral projections of sensory fascicles in the human superficial radial nerve.

The sensory territories of different cutaneous fascicles of the superficial radial nerve were delineated by microneurography at the level of the distal forearm in humans. Three fascicular patterns were found at this level: one supplying the dorsum of the radial aspect of the dorsum of the hand over the first dorsal interosseous space; another supplying the lateral aspect of the first metacarpal extending to the lateral aspect of the thumb; and a third innervating the second interosseous space and the proximal phalanx of the index and middle fingers. The compound fascicular territory is comparable to the classical territory described for the superficial radial nerve. Intraneural microstimulation of individual fascicles did not evoke paraesthesiae or pain beyond their fascicular territory, regardless of the stimulus intensity. We conclude that the superficial radial nerve at the forearm in man is composed of only three fascicles, as shown by the present study and from previous anatomical work. Referred pain seems related to nerve activity in afferent fibres from fascicles supplying deep tissues and muscles, not from cutaneous afferents.

Synaptic contacts established by inferior alveolar nerve fibres in the paratrigeminal nucleus: an electron microscopic study in the rat.

A quantitative evaluation of the types of synaptic contacts from afferent fibres in the paratrigeminal nucleus after partial pulpectomy was compared with that after transection of the inferior alveolar nerve (IAN), using transganglionic degeneration. Degenerating terminals with a marked increase in axoplasmic electron opacity were observed bilaterally in the paratrigeminal nucleus of rats submitted to either partial pulpectomy or IAN transection. The total number of degenerating terminals observed after partial pulpectomy was 53% of that for IAN transection. This suggests a considerable contribution of tooth pulp afferent fibres in the total number of synaptic contacts in the intermediate and caudal parts of the paratrigeminal nucleus. In both the partial pulpectomy and IAN-transected groups, the majority of these synapses formed single asymmetric contacts with intermediate and distal dendritic segments, and accounted for 74% of all classified contacts. The remaining 26% of contacts occurred with proximal dendritic segments, dendritic spines, perikaryon, normal terminals and double post-synaptic elements. There was no statistically significant difference in the number of synaptic contacts for each type of synapse, with the exception of contacts with dendritic spines in the contralateral side, between the partially pulpectomised and IAN-transected groups.

[Normal pattern of intraepidermal nerve fibers in 30 healthy volunteers with PGP 9.5]. / Padronização normal das fibras nervosas intraepidérmicas em 30 voluntários saudáveis com PGP 9,5.

Skin biopsy has become an attractive technique to evaluate the terminal regions of small nerve fibers. There is extensive innervation of the skin by both sensory and autonomic fibers as demonstrated by staining for the pan-axonal marker PGP 9,5. The normal pattern is fundamental before any study, since three different techniques described in the literature with different results. Skin biopsy specimens of 3-mm in diameter were obtained from the distal leg of 30 healthy controls. Median intraepidermal nerve fiber density was 5.3/mm. Skin biopsy may be a useful tool for assessing the topographic extent and degree of nerve fiber damage in sensory neuropathies and may be particularly useful in experimental treatment trials for peripheral neuropathies since, in contrast to standard nerve biopsy, the test can be repeated.

Padronização normal das fibras nervosas intraepidérmicas em 30 voluntários saudáveis com PGP 9, 5 / Normal pattern of intraepidermal nerve fibers in 30 healthy volunteers with PGP 9.5

O recente método de avaliação das fibras nervosas intraepidérmicas com o PGP 9,5 vem se mostrando de grande utilidade no diagnóstico das neuropatias sensitivas de fibras finas, autonômicas e neuropatias periféricas subclínicas. Devido à variação da técnica relatada na literatura é de fundamental importância uma padronização normal. Estudamos 15 homens e 15 mulheres com média de idade de 34,5 anos. Em todos os voluntários foi realizada biopsia de pele na porção distal da perna. A média da densidade linear das fibras nervosas intraepidérmicas foi 5,3/mm com mediana de 6,0 e desvio padrão de 1,94. Essa técnica possui um grande número de vantagens em relação à biopsia de nervo convencional, é simples, pouco invasiva, reproduzível e pode ser repetida no mesmo paciente para avaliar progressão da neuropatia e possíveis respostas terapêuticas.

Tridimensional architecture of the collagen element in the arachnoid granulations in humans: a study on scanning electron microscopy.

The arachnoid granulations of adult individual of both sexes were studied through scanning electron microscopy. The dura mater and arachnoid meninges of individuals were collected at the Service of Death Verification of São Paulo - USP and fixed in Karnovsky solution. After this period the material was prepared for analysis in electron microscope. Our results demonstrated that the arachnoid granulations are formed by a pedicle, body and apex, being surrounded by a capsule of connective tissue, which in turn is composed of, basically, bundles of collagen fibers that line pores of different shapes and sizes. The smaller pores are lined by tiny bundles and are located at the apical region of the granulation and the larger are lined by thicker bundles and are located at the lateral regions. In the body we verified that the bundles of collagen fibers compose a fibrous meshwork and in some regions these bundles have circular orientation, forming pores similar to those found at the region of the capsule.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane.

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.