The course and distribution of the buccal nerve: clinical relevance in dentistry.

BACKGROUND: The buccal branch of the mandibular division of the trigeminal nerve is commonly anaesthetized for dental procedures and may be damaged during surgery. Descriptions of the distribution of the buccal nerve (BN) in anatomical texts are generally lacking in detail and do not provide information about the extent of its variation between individuals. There are also commonly-held clinical beliefs about the BN that lack support from anatomical dissections. METHODS: Detailed dissections of the course and distribution of the BN were performed in a sample of 12 hemi-heads from 11 edentulous and partially dentate human adult cadavers. RESULTS: A broader distribution of the BN was found than described previously, with innervation extending to the lips in all cases. Approximately half of the lateral sides of the lips were innervated by the BN in two cases and approximately one-third of their lateral sides in the other 10 cases. Distribution of the BN to the lower lips was wider than to the upper lips. CONCLUSIONS: Our findings provide a stronger anatomical basis to underpin clinical procedures involving the BN and indicate that some commonly-held clinical views about this nerve are not supported by anatomical evidence.

The human medial pterygoid muscle: Attachments and distribution of muscle spindles.

Published descriptions about the sites of origin of the human medial pterygoid muscle vary and there are few reports on the distribution and density of muscle spindles in this muscle. We aimed to: (1) determine the extent of anatomical variability in the origins and insertions of the superficial and deep heads of the human medial pterygoid muscle and (2) determine the extent of variation in the distribution of spindles in the two heads of the muscle. Thirty-nine human cadaver hemi-heads were dissected and the attachments of the medial pterygoid muscle examined. The whole muscle was removed, weighed, cut into segments and embedded in wax for light microscopy. Sections were stained with Weigert-Van Gieson stain and scanned into digital images. Spindles were manually counted. In seven specimens, the deep head of the muscle arose from the medial surface of the lateral pterygoid plate and the pterygoid fossa. In 28 specimens, the origin extended onto the lateral surface of the medial pterygoid plate. There were abundant muscle spindles in the middle of the muscle, slightly fewer 1 cm toward the insertion, significantly fewer 1 cm toward the origin, and few or no spindles near the origins of the superficial and deep heads or near their insertion. In conclusion, firstly, this study shows that in 72% of the specimens examined, the origin of the medial pterygoid is wider than conventionally described in anatomical textbooks. Secondly, the segmental distribution of muscle spindles is described for the first time. Clin. Anat. 30:1064-1071, 2017. © 2017 Wiley Periodicals, Inc.

An unusual ocular complication following dental anaesthesia: case report.

Used routinely by dental practitioners, local anaesthetics are generally a safe and effective means of achieving pain control during invasive dental procedures. Delivery, however, is technique sensitive and the potential for patient complication exists. Although reasonably rare, ocular complications have occurred, often leaving the patient and the clinician in distress. Such reported events have almost always involved tissue responses ipsilateral to the injection site. The current case report presents an unusual event involving involuntary fasciculation or hemifacial spasm and eventual eyelid closure on the contralateral side following a routine inferior alveolar nerve block. Aspects including the patient's history, the observed response, a pathophysiological hypothesis and patient management are discussed.

Under your nose: a rare finding during dissection provides insights into maxillary supernumerary teeth.

BACKGROUND: A supernumerary tooth was found during anatomical dissection. The position of this tooth, still impacted in the maxilla, and the associated pathology make this a rare case. METHODS: During dissection by dental students of the sagittally-sectioned head of a cadaver, a supernumerary tooth was identified in the mid-palatal area. Further dissection revealed a swelling with a thin bony covering related to the crown of the tooth. The maxilla was removed en bloc and radiographic examination, CT scanning, electron microscopy and histology were undertaken. RESULTS: The tooth had a crenulated occlusal surface and a single root. It was 25 mm posterior to the root apex of the permanent upper central incisor. The swelling, confirmed by radiographs and CT imaging to be associated with the crown, occupied approximately one-third of the maxillary sinus. The 3D shape of the cystic lesion was visualized by a composite digital movie. CONCLUSIONS: The crown form, position of the tooth and the associated dentigerous cyst suggested it was a palatally developing supernumerary premolar which had been displaced to the palatal midline by the expanding cyst. This rare case highlights the learning and teaching opportunities available during dissection, showing important variations in both development and clinical anatomy.

Evaluating the role of substance P in the growth of brain tumors.

Recent research has investigated the expression and secretion of neuropeptides by tumors, and the potential of these peptides to facilitate tumor growth and spread. In particular, substance P (SP) and its receptor NK1 have been implicated in tumor cell growth and evasion of apoptosis, although few studies have examined this relationship in vivo. The present study used both in vitro and in vivo models to characterize the role of SP in tumor pathogenesis. Immunohistochemical assessment of human primary and secondary brain tumor tissue demonstrated a marked increase in SP and its NK1 receptor in all tumor types investigated. Of the metastatic tumors, melanoma demonstrated particularly elevated SP and NK1 receptor staining. Subsequently, A-375 human melanoma cell line was examined in vitro and found to express both SP and the NK1 receptor. Treatment with the NK1 receptor antagonist Emend IV resulted in decreased cell viability and an increase in cell death in this cell line in vitro. An animal model of brain tumors using the same cell line was employed to assess the effect of Emend IV on tumor growth in vivo. Administration of Emend IV was found to decrease tumor volume and decrease cellular proliferation indicating that SP may play a role in tumor pathogenesis within the brain. We conclude that SP may provide a novel therapeutic target in the treatment of certain types of brain tumors, with further research required to determine whether the role of SP in cancer is tumor-type dependent.

Applied anatomy of the pterygomandibular space: improving the success of inferior alveolar nerve blocks.

A thorough knowledge of the anatomy of the pterygomandibular space is essential for the successful administration of the inferior alveolar nerve block. In addition to the inferior alveolar and lingual nerves, other structures in this space are of particular significance for local anaesthesia, including the inferior alveolar vessels, the sphenomandibular ligament and the interpterygoid fascia. These structures can all potentially have an impact on the effectiveness of local anaesthesia in this area. Greater understanding of the nature and extent of variation in intraoral landmarks and underlying structures should lead to improved success rates, and provide safer and more effective anaesthesia. The direct technique for the inferior alveolar nerve block is used frequently by most clinicians in Australia and this review evaluates its anatomical rationale and provides possible explanations for anaesthetic failures.

Magnesium restores altered aquaporin-4 immunoreactivity following traumatic brain injury to a pre-injury state.

Magnesium reduces edema following traumatic brain injury (TBI), although the associated mechanisms are unknown. Recent studies suggest that edema formation after TBI may be related to alterations in aquaporin-4 (AQP4) channels. In this study, we characterize the effects of magnesium administration on AQP4 immunoreactivity following TBI. Male Sprague-Dawley rats were injured by impact-acceleration diffuse TBI and a subgroup was administered 30 mg/kg magnesium sulphate 30 minutes after injury. Animals were fixed by perfusion 5 hours later, which corresponded to the time of maximum edema formation according to previous studies. One half of the brain was cut using a Vibratome and the other half blocked in paraffin wax. Wax and Vibratome sections were immunostained for detection of AQP4 by light and electron microscopy, respectively. In untreated animals, AQP4 immunoreactivity was increased in the subependymal inner glia limitans and the subpial outer glia limitans, and decreased in perivascular astrocytic processes in the cerebrum and brain stem. In contrast, animals treated with magnesium sulphate had AQP4 profiles similar to normal and sham control animals. We conclude that magnesium decreases brain edema formation after TBI, possibly by restoring the polarized state of astrocytes and by down-regulation of AQP4 channels in astrocytes.

Paranodal pathology in Tangier disease with remitting-relapsing multifocal neuropathy.

Pathological studies of a sural nerve biopsy in a man with Tangier disease presenting as a remitting-relapsing multifocal neuropathy showed abnormalities in the paranodal regions, including lipid deposition (65%) and redundant myelin foldings, with various degrees of myelin splitting and vesiculation (43%) forming small tomacula and abnormal myelin terminal loops (4%). The internodal regions were normal in the majority of myelinated fibres. Abnormal lipid storage was also present in the Schwann cells of the majority of unmyelinated fibres (67%). The evidence suggests that the noncompacted myelin region of the paranode is a preferential site for lipid storage in the myelinated Schwann cell, and that the space-occupying effects of the cholesterol esters leads to paranodal malfunction and tomacula formation as the pathological basis for the multifocal relapsing-remitting clinical course.

Early paranodal myelin swellings (tomacula) in an avian riboflavin deficiency model of demyelinating neuropathy.

INTRODUCTION: Disruption of the complex architectural and molecular organization of the paranodal region of myelinated peripheral nerve fiber may initiate the evolving time dependent process of segmental demyelination. In support of this notion was the finding of focal paranodal myelin swellings (tomacula) due to redundant folding of myelin sheaths, early in the time course of an avian riboflavin deficiency model of demyelinating neuropathy. METHODS: Newborn broiler meat chickens were maintained either on a routine diet containing 5.0 mg/kg riboflavin (control group) or a riboflavin-deficient diet containing 1.8 mg/kg riboflavin. Riboflavin concentrations in the liver were measured at postnatal day 11. Peripheral nerves were morphologically examined at days 6, 11, 16 and 21 using light and electron microscopy and teased nerve fiber techniques. RESULTS: Riboflavin-deficient chickens showed signs of a neuropathy from days 8 and pathological examination of peripheral nerves revealed a demyelinating neuropathy with paranodal tomacula formation starting on day 11. Paranodal tomacula consisted of redundant myelin infoldings or outfoldings, increased in size and frequency after day 11. After day 16, the paranodal swellings showed prominent degenerative changes accompanied by an increased frequency of myelinated fibers showing demyelination. CONCLUSION: Tomacula due to redundant myelin folds are generally considered a remyelination phenomenon, yet in this avian riboflavin deficiency model of demyelination, the paranodal tomacula occurred early in the course of demyelination.

Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glands.

Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague-Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels.

A narrow time-window for access to the brain by exogenous protein after immunological targeting of a blood-brain barrier antigen.

The endothelial barrier antigen (EBA) is a membrane protein expressed by endothelial cells of the rat blood-brain barrier (BBB). A previous short-term non-recovery study demonstrated that immunological targeting of EBA by intravenous administration of a monoclonal antibody (anti-EBA) led to acute opening of the BBB to exogenous and endogenous tracers. The aims of the present study were to determine whether opening of the BBB was reversible and compatible with survival, and whether a "therapeutic window" existed. A single intravenous injection of one of three doses (high, medium and low) of anti-EBA was used. Animals were allowed to survive for periods ranging from 17 min to 4 days. The tracer horseradish peroxidase (HRP) was administered intravenously 10 min before perfusion fixation, and its distribution was assessed in Vibratome sections of the brain and spinal cord. Leakage of HRP into the central nervous system was dose- and time-dependent. The medium dose produced incipient HRP leakage at 17 min and widespread pronounced leakage at 30 min. Progressive reduction in HRP permeability occurred from 45 min to 2 h, with barrier restoration by 3 h. At all subsequent time intervals (6 h-4 days) the BBB remained impermeable to HRP. The low and high doses produced less and greater HRP leakage, respectively, but restoration of the barrier still occurred at 3 h. The high dose, however, produced a number of deaths. Animals treated with an isotype control antibody showed no HRP leakage at comparable time intervals. The results indicated that (1) this model was compatible with survival, (2) opening of the BBB was monophasic and transient, occurring during a narrow "time-window", and (3) the barrier, once reconstituted, maintained its integrity.

Expression of a blood-brain barrier-specific antigen in the reproductive tract of the male rat.

The endothelial barrier antigen (EBA) is a protein expressed specifically by the endothelial cells of the rat brain barrier vessels. This antigen has been described as a 'barrier protein' and is used as a marker for the competent blood-brain barrier. A blood-testis barrier has also been described. However, unlike the blood-brain barrier, which is formed by endothelial cells, the blood-testis barrier is formed mainly by the Sertoli cells, which provide an isolated environment for spermatogenic cells within the seminiferous tubules. Testicular blood vessels express the erythroid glucose transporter protein and other markers, which are strongly expressed in brain blood vessels, and may contribute to the blood-testis barrier. This study was carried out to determine whether Sertoli cells or testicular blood vessels express EBA. Tissues of other organs were used as controls for EBA expression. EBA was expressed by the endothelial cells in most microvessels of the testis, and in a few vessels of the epididymis, seminal vesicle, prostate gland, vas deferens and bladder-neck region. Furthermore, EBA was strongly and consistently detected in epithelial cells of the rete testis and dorsolateral prostate gland, and in a few epithelial cells of the ventral prostate gland, the seminal vesicle and the coagulating gland. However, Sertoli cells, which are the main site of the blood-testis barrier, were negative for EBA. In conclusion, EBA may have a wider role in rat tissues than has been previously appreciated.

Clostridium perfringens prototoxin-induced alteration of endothelial barrier antigen (EBA) immunoreactivity at the blood-brain barrier (BBB).

It has been reported that the severe cerebral edema produced in experimental animals by Clostridium perfringens (Cl p) type D epsilon toxin can be prevented by prior treatment with its precursor prototoxin due to competitive binding to endothelial cells (ECs) at the blood-brain barrier (BBB). In this study we investigate the effects of the prototoxin on the BBB, without added toxin. The integrity of the BBB was assessed by its ability to prevent leakage of endogenous albumin. ECs at the BBB were studied by immunocytochemistry for any alteration in the endothelial barrier antigen (EBA), a molecular marker for the intact BBB. Immunocytochemistry showed rapid but mild opening of the BBB to endogenous albumin. Light and electron immunocytochemistry showed qualitative and quantitative reduction in EBA immunoreactivity, with a spectrum of changes at time intervals from 1 h to 14 days post-prototoxin injection. Some vessels with ultrastructural changes and widening of the perivascular space retained EBA immunoreactivity. Many vessels showed partial or complete loss of EBA staining with minimal widening of the perivascular space and edema. Recovery of EBA expression was still incomplete at 14 days postinjection. This is the first report to show endothelial cell damage, mild reversible cerebral edema, and alteration in BBB markers following administration of Cl p prototoxin. This model of mild brain edema may be useful for BBB studies.

Immunological targeting of the endothelial barrier antigen (EBA) in vivo leads to opening of the blood-brain barrier.

The role of the endothelial barrier antigen (EBA) in the blood-brain barrier (BBB) of the rat is not fully understood. Pathological conditions which show BBB disruption and leakage of plasma proteins are associated with reduced EBA expression in brain endothelial cells (ECs). However, it is not known if the reduction in EBA is the primary event or is secondary to protein extravasation. We hypothesized that immunological targeting of EBA in vivo would lead to opening of the BBB. To test this hypothesis, a monoclonal antibody (anti-EBA) was intravenously injected in anaesthetized experimental rats. Control animals received intravenous injections of phosphate-buffered saline (PBS) or non-specific antibodies (anti-human cytokeratin, anti-Salmonella bacterial antigen, or anti-pan endothelial cell antigen). Two groups of rats were used, each included experimental and control animals. The first group was used for immunocytochemical detection of EBA in brain ECs and rat albumin in brain parenchyma. In the second group, the permeability of the BBB to horseradish peroxidase (HRP) was tested. Experimental animals, injected with anti-EBA antibody, showed extensive leakage of HRP and albumin in the grey and white matter of the brain. Immunocytochemistry of experimental brains showed that the intravenously injected anti-EBA became bound to ECs and was detected in tissue sections. Control animals did not show leakage of HRP or albumin, and EBA distribution was normal. This study demonstrated for the first time, that immunological 'neutralisation' of EBA leads to opening of the BBB, and provided direct evidence for the importance of EBA in maintaining the integrity of the BBB in the rat.

Toxin-induced vasogenic cerebral oedema in a rat model.

Vasogenic cerebral oedema (VCO) was induced in Hooded Wistar rats by intraperitoneal injection of Clostridium perfringens type D epsilon prototoxin. Animals were killed, 1 h to 14 d postinjection, by perfusion fixation under general anaesthesia. VCO was detected by the presence of endogenous albumin in the brain, visualised by immunocytochemistry. As early as 1 h postinjection, albumin was detected in the walls of cerebral microvessels. Maximal diffuse leakage within the neural parenchyma was seen at 24 and 48 h and immunoreactivity was still present at 4 d. At 7 d only few foci were seen, and at 14 d albumin distribution was similar to that in controls. Ultrastructural assessment of the microvessels showed swelling of many astrocytic processes and abnormalities of the endothelial cells varying from swelling with loss of cytoplasmic organelles to cells showing increased electron density. Immunostaining for the endothelial barrier antigen (EBA) showed strongly immunoreactive vessels throughout normal brains. Experimental animals showed partial reduction in EBA expression, most evident at 24 and 48 h, with gradual recovery to normal by 14 d. The exact role that EBA plays in the intact BBB remains obscure.

Substance P-induced enhanced permeability of dura mater microvessels is accompanied by pronounced ultrastructural changes, but is not dependent on the density of endothelial cell anionic sites.

Experimental data point to a determinant role for endothelial cell (EC) anionic sites in the regulation of vascular permeability. Previous studies have shown that EC anionic sites density is reduced in conditions of enhanced permeability. The pathophysiology of migraine and vascular headache encompasses dilatation of dural vessels and extravasation of plasma proteins. The current study was carried out to determine if the density of EC anionic sites is reduced in enhanced permeability of dural vessels. Enhanced permeability was chemically induced in rats by intravenous injection of substance P and was tested by assessing leakage of horseradish peroxidase (HRP). Anionic sites were labelled with cationic colloidal gold and their density was quantified from electron microscopy negatives. Experimental animals showed increased leakage of HRP from dural vessels. However, anionic sites in EC membranes (luminal and abluminal) showed no statistical differences when their mean densities in experimental and control animals were compared. The results indicate that in this model, factors other than the density of anionic sites may be important determinants in the permeability of dural vessels. Such factors may include structural alteration of ECs consistent with an increased permeability. In this study pronounced ultrastructural changes in ECs were noted in experimental animals including widening of intercellular junctions and an increase in the number of EC gaps and vesicles.

Dural microvessels: molecular properties of their luminal anionic sites.

(1) Neurogenic inflammation has been implicated in the pathogenesis of the vascular headaches of migraine and cluster headaches. (2) Dural blood vessels are both pain-sensitive and show neurogenic plasma extravasation. (3) Endothelial cell (EC) surface anionic sites appear to be a determinant of vascular permeability. We therefore examined the anionic sites of dural EC to determine whether they are different from those of pial and parenchymal vessels. Luminal anionic sites of rat optic nerve EC were labelled with cationic colloidal gold (CCG) and cationic ferritin (CF) and examined by electron microscopy. Employing a battery of enzymes, the effects of digestion of ultrathin sections on subsequent labelling with CCG was quantified using image analysis software. In addition, a gold-labelled lectin, wheat-germ agglutinin (WGA), was employed to locate specific saccharide residues. Of the enzymes with a narrow specificity, only neuraminidase substantially reduced CCG binding. Of the proteolytic enzymes, papain was most effective in reducing labelling. These results show that the luminal EC anionic sites are chiefly composed of sialoglycoproteins. The labelling with biotinylated WGA-streptavidin gold was similar to that with CCG without enzyme digestion. This suggests that WGA is binding to N-acetylneuraminic (sialic) acid residues and not to the neutral N-acetylglucosamine (since CCG would not label uncharged molecules). These results do not differ significantly from those for pial and parenchymal EC. It is therefore likely that factors other than anionic site molecular composition account for the susceptibility of dural vessels to neurogenic plasma extravasation. The relevance of these observations in an experimental animal model to the human clinical condition remains to be determined.

Optic nerve microvessels: a partial molecular definition of cell surface anionic sites.

Incorporated in the luminal glycocalyx of vascular endothelia (EC) are negatively charged microdomains (anionic sites). These sites are considered functionally important (a) in their interaction with circulating blood constituents, and (b) as a determinant of vascular permeability. The molecular composition of these EC sites, described for a number of tissues, has demonstrated a heterogeneity dependent on their anatomical location. Luminal anionic sites have not been characterized for EC of optic nerve. Optic nerves were removed from Sprague-Dawley rats previously fixed by vascular perfusion. EC anionic sites were labelled with the probes cationic colloidal gold (CCG) and cationic ferritin (CF), using the pre- and post-embedding techniques, and examined by electron microscopy. The effects of enzyme digestion of ultrathin sections on subsequent CCG labelling were determined using a battery of enzymes in association with the post-embedding technique. CCG labelling was quantified following each enzyme treatment using image analysis software. The biotinylated lectin wheat germ agglutinin (WGA) with streptavidin gold was also used to localize specific monosaccharide residues. The luminal front of intraneural EC showed a uniform labelling with CCG and CF which was greater than on the abluminal surface. Extracellular matrix components and basal laminae were moderately labelled. Digestion of tissue sections with heparitinase and trypsin had no significant effect on subsequent CCG labelling. Proteinase K was less effective than papain but both produced a significant reduction. Neuraminidase almost completely eliminated labelling. CCG binding to the luminal plasma membrane of optic nerve EC can be significantly reduced with proteolytic and glycolytic enzymes. The results demonstrate that sialoglycoproteins principally constitute these luminal EC anionic sites. Biotinylated WGA-streptavidin gold, which detects both N-acetylneuraminic (sialic) acid and N-acetylglucosamine, gave a similar pattern of labelling to CCG alone on the luminal versus abluminal EC fronts. These findings suggest that WGA is binding predominantly to N-acetylneuraminic acid residues since CCG would not label the neutral (uncharged) N-acetylglucosamine.

Distribution of a putative endothelial barrier antigen in the ocular and orbital tissues of the rat.

BACKGROUND: A rat endothelial barrier antigen (EBA) recognised by a monoclonal antibody has been shown to be expressed strongly by endothelial cells of brain capillaries possessing a blood-brain barrier and only weakly expressed by fenestrated brain vessels. METHODS: In this study immunocytochemical methods for light and electron microscopy were used to study EBA distribution in the eye and orbital tissues of the rat. RESULTS: Blood-ocular barrier vessels in the optic nerve, retina, iris, and some vessels in th choroid and ciliary body were immunopositive for EBA. By pre-embedding immunocytochemistry for electron microscopy the antigen was observed on the luminal endothelial cell surface. CONCLUSION: Surprisingly, some non-barrier vessels in the ciliary body and choroid expressed EBA suggesting that it may play a broader role in endothelial properties than previously recognised. The functional significance of EBA remains to be elucidated.

Differential expression of an endothelial barrier antigen between the CNS and the PNS.

A monoclonal antibody to an antigen (EBA) expressed by neural endothelial cells (EC) was used to investigate any difference in the distribution of EBA between the CNS and PNS. Pre-embedding ultrastructural cytochemistry of rat sciatic and optic nerves was undertaken using anti-EBA, detected with a silver-enhanced gold-conjugated secondary antibody. LM immunocytochemical localisation of EBA was also performed using an HRP-conjugated secondary antibody. EC of pial and parenchymal optic nerve vessels were strongly immunopositive for EBA. Vessels of the dura were negative. At the EM level EBA was observed on the EC luminal surface. In contrast, EC of sciatic nerve were either negative or only weakly immunopositive. The molecular characteristics and function of EBA are largely unknown. Therefore the functional significance of the present findings remains to be determined.