Anatomy of the mastoid triangle and morphometric sex differences.

OBJECTIVE OF THE STUDY: This study aimed to quantify the area of the mastoid triangle (MT) and assess potential morphometric differences between males and females. PATIENTS: The sample consisted of 244 dry human skulls, with biological sex known based on genetic analysis, collected from a medicolegal osteological database from Central-Western Brazil. MATERIALS AND METHODS: The study was observational, analytical, and cross-sectional. The skulls were analyzed using Heron's equation to calculate the area of the MT. The landmarks connecting each of the sides of the triangle were: Porion (Po)>Mastoidale (Ma)>Asterion (Ast). Morphometric references were calculated and compared based on sex. RESULTS: The area of the MT was nearly 14% larger in males compared to females (p<0.05). The mean MT area for the right and left sides of males were 684.11±93.25mm2 and 668.94±111.95mm2, respectively. In females, the mean MT for the right and left sides were 588.93±91.09mm2 and 582.88±102.98mm2, respectively. Right and left side measurements were significantly different (p<0.05), except for Po-Ast (p=0.232). CONCLUSION: Morphometric features regarding the MT were slightly different between males and females. Application of the MT as a dimorphic tool should be adjuvant. Moreover, this tool should be considered carefully, especially because the sex-based differences were statistically significant, but discrete between males and females.

Influence of the Flowable Resin Layer on Bond Strength Between Resin Cement and a Universal Adhesive Applied in the Immediate Dentin-sealing Technique.

OBJECTIVES: The present study evaluated the influence of a flowable resin layer on bond strength between resin cement and a universal adhesive applied using an immediate dentin sealing (IDS) technique. METHODS AND MATERIALS: Coronary portions of bovine teeth were randomly divided into six groups (n=15). In the IDS.U group, the exposed dentin was immediately sealed with the Single Bond Universal adhesive (3M ESPE) following the self-etching protocol. In the IDS.UF group, a layer of Filtek Z350 (3M ESPE) flow resin was applied over the universal adhesive. In the DDS (control) group, the dentin was kept "fresh" and delayed dentin sealing was performed. After 24 hours in distilled water at 37°C, dentin surfaces were treated with pumice, phosphoric acid, and the application of the universal adhesive in the IDS.U and IDS. UF groups. The DDS group was treated with pumice and the universal adhesive was applied. The samples received cylinders of resin cement Rely X Ultimate (3M ESPE) made with the aid of starch tubes of 0.96 mm in diameter and 2 mm in length. They were submitted to the microshear bond strength test (µSBS) at 0.5 mm/min, after 24 hours (T1) and 3 months (T2). The fracture areas were evaluated qualitatively using a DSM 300 microscope (KOZO) with 45× magnification and classified as: adhesive, cohesive in cement, cohesive in dentin, or mixed. Samples were analyzed by scanning electron microscopy (SEM). The data were compared statistically between groups using the Kruskal-Wallis test, and intra-groups using the Mann-Whitney test (α=0.05). RESULTS: There were no significant differences between groups for the bond strength values (p>0.05). The IDS.UF group showed higher values at 3 months, when compared to the values of 24 hours (p<0.001). All groups showed a predominance of adhesive fracture (86.7% to 100%). SEM showed dentinal tubules exposed in the IDS.U and DDS groups; in the IDS.UF group, the tubules were completely sealed. CONCLUSIONS: The flow resin can be used on the adhesive when using the IDS technique because it increased the bond strength values after 3 months and promoted effective sealing of the dentinal tubules.

Reliability and validity of two software systems used to measure the pharyngeal airway space in three-dimensional analysis.

The aim of this study was to test the reliability and validity of two software systems used to measure the pharyngeal airway space three-dimensionally. A sample of 40 cone beam computed tomography images from adult patients was taken from a database. The cone beam computed tomography images were analysed by InVivoDental and Dolphin 3D software systems by two calibrated examiners. Three nasopharynx and oropharynx prototypes were used as a reference standard to validate the software systems. The volume, minimum area and minimum area localization were the measurements tested. Measurements were compared using a paired t-test; correlated using Pearson's correlation and linear regression. Bland-Altman analysis was also used. We found significant differences in the oropharynx volume (P=0.002) and nasopharynx minimum area localization (P=0.009). The Dolphin 3D software presented higher-volume values than the ones found in the prototype, while the InVivoDental software presented lower values. Strong (r>0.7; P>0.001) or very strong (r>0.9; P>0.001) correlations were observed between the software systems. Bland-Altman analysis found good agreement between prototypes and the software systems. The measurements obtained from the Dolphin 3D and InVivoDental software systems are both reliable, strongly correlated, but should not be assumed as equal. Dolphin 3D software overestimates the nasopharynx and oropharynx volumes, while the InVivoDental software underestimates them.

Amelioration of non-motor dysfunctions after transplantation of human dopamine neurons in a model of Parkinson's disease.

BACKGROUND: Patients suffering from Parkinson's disease (PD) display cognitive and neuropsychiatric dysfunctions, especially with disease progression. Although these impairments have been reported to impact more heavily upon a patient's quality of life than any motor dysfunctions, there are currently no interventions capable of adequately targeting these non-motor deficits. OBJECTIVES: Utilizing a rodent model of PD, we investigated whether cell replacement therapy, using intrastriatal transplants of human-derived ventral mesencephalic (hVM) grafts, could alleviate cognitive and neuropsychiatric, as well as motor, dysfunctions. METHODS: Rats with unilateral 6-hydroxydopamine lesions to the medial forebrain bundle were tested on a complex operant task that dissociates motivational, visuospatial and motor impairments sensitive to the loss of dopamine. A subset of lesioned rats received intrastriatal hVM grafts of ~9 weeks gestation. Post-graft, rats underwent repeated drug-induced rotation tests and were tested on two versions of the complex operant task, before post-mortem analysis of the hVM tissue grafts. RESULTS: Post-graft behavioural testing revealed that hVM grafts improved non-motor aspects of task performance, specifically visuospatial function and motivational processing, as well as alleviating motor dysfunctions. CONCLUSIONS: We report the first evidence of human VM cell grafts alleviating both non-motor and motor dysfunctions in an animal model of PD. This intervention, therefore, is the first to improve cognitive and neuropsychiatric symptoms long-term in a model of PD.

Increased efficacy of the 6-hydroxydopamine lesion of the median forebrain bundle in small rats, by modification of the stereotaxic coordinates.

The 6-hydroxydopamine (6-OHDA) lesion is the most widely used rat model of Parkinson's disease. A single unilateral injection of 6-OHDA into the median forebrain bundle (MFB) selectively destroys dopamine neurons in the ipsilateral substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), removing more than 95% of the dopamine innervation from target areas. The stereotaxic coordinates used to deliver 6-OHDA to the MFB have been used in our laboratory successfully for more than 25 years. However, in recent years we have observed a decline in the success rate of this lesion. Previously regular success rates of >80% of rats lesioned, have become progressively more variable, with rates as low as 20% recorded in some experiments. Having excluded variability of the neurotoxin and operator errors, we hypothesized that the change seen might be due to the use of smaller rats at the time of first surgery. An attempt to proportionally adjust the lesion coordinates base on head size did not increase lesion efficacy. However, in support of the small rat hypothesis it was observed that, using the standard coordinates, rat's heads had a "nose-up" position in the stereotaxic fame. Adjustment of the nose bar to obtain a flat head position during surgery improved lesion success, and subsequent adjustments of the lesion coordinates to account for smaller head size led to a greatly increased lesion efficacy (>90%) as assessed by amphetamine induced rotation.

Medical terminations of pregnancy: a viable source of tissue for cell replacement therapy for neurodegenerative disorders.

"Proof-of-principle" that cell replacement therapy works for neurodegeneration has been reported, but only using donor cells collected from fetal brain tissue obtained from surgical terminations of pregnancy. Surgical terminations of pregnancy represent an increasingly limited supply of donor cells due to the tendency towards performing medical termination in much of Europe. This imposes a severe constraint on further experimental and clinical cell transplantation research. Therefore, we explore here the feasibility of using medical termination tissue as a donor source. Products of conception were retrieved from surgical terminations over the last 7 years and from medical terminations over the last 2.5 years. The number of collections that yielded fetal tissue, viable brain tissue, and identifiable brain regions (ganglionic eminence, ventral mesencephalon, and neocortex) were recorded. We studied cell viability, cell physiological properties, and differentiation potential both in vitro and following transplantation into the central nervous system of rodent models of neurodegenerative disease. Within equivalent periods, we were able to collect substantially greater numbers of fetal remains from medical than from surgical terminations of pregnancy, and the medical terminations yielded a much higher proportion of identifiable and dissectible brain tissue. Furthermore, we demonstrate that harvested cells retain the capacity to differentiate into neurons with characteristics appropriate to the region from which they are dissected. We show that, contrary to widespread assumption, medical termination of pregnancy-derived fetal brain cells represent a feasible and more readily available source of human fetal tissue for experimental cell transplantation with the potential for use in future clinical trials in human neurodegenerative disease.

Thoughts on aneuploidy.

Aneuploidy refers to karyotypic abnormalities characterized by gain or loss of individual chromosomes. This condition is associated with disease and death in all organisms in which it has been studied. We have characterized the effects of aneuploidy on yeast and primary mouse cells and found it to be detrimental at the cellular level. Furthermore, we find that aneuploid cells exhibit phenotypes consistent with increased energy need and proteotoxic stress. These observations, together with the finding that the additional chromosomes found in aneuploid cells are active, lead us to propose that aneuploidy causes an increased burden on protein synthesis and protein quality-control pathways and so induces an aneuploidy stress response.

A simple breeding protocol for the procurement of accurately staged rat donor embryos for neural transplantation.

Obtaining accurately staged rat embryos can be difficult because of the variety of breeding protocols employed and because precise staging cannot be confirmed until excision of the embryos from the dam. The detection of estrus, pairing of animals, and confirmation of pregnancies is generally left to commercial suppliers, as in-house breeding can be laborious and unpredictable. Here we describe a simple, reliable in-house breeding protocol for the generation of accurately staged embryos as assessed by measurements of average crown to rump length (CRL).

A rat embryo staging scale for the generation of donor tissue for neural transplantation.

In rat models of Parkinson's and Huntington's diseases, embryonic neural cells obtained from embryos of specified ages can be implanted into the brain to partially restore both physiology and function. However, in litters produced using overnight mating protocols (often from commercial suppliers), the embryonic age can be difficult to determine precisely. As a result, embryonic size based on crown to rump length (CRL) is usually a more reliable method of embryo staging than the day of mating. This approach is not without difficulty. There are a number of rat staging scales in the literature, none of which deal with donor ages younger than E13, and there are discrepancies between scales at some donor ages. In the present article, we have devised a short mating-period protocol to produce precisely aged embryos. We show that CRL is a highly accurate, reproducible index of donor age and we present an updated embryonic staging scale for Sprague-Dawley (CD) rats that includes donor ages younger than those previously reported.

Recovery of functional deficits following early donor age ventral mesencephalic grafts in a rat model of Parkinson's disease.

It has previously been reported that dopaminergic grafts derived from early donor age, embryonic age 12-day-old (E12) rat embryos produced a fivefold greater yield of dopamine neurons than those derived from conventional E14 donors. The present study addresses whether E12 grafts are able to ameliorate lesion-induced behavioral deficits to the same extent as E14 grafts. In a unilateral rat model of Parkinson's disease, animals received grafts derived from either E12 or E14 donor embryos, dispersed at four sites in the lesioned striatum. Both E12 and E14 grafts were able to induce recovery on both amphetamine and apomorphine rotation tests, and to ameliorate deficits in the cylinder, stepping test, and corridor tests, but were unable to restore function in the paw reaching task. E12 grafts were equivalent to E14 grafts in their effects on lesion-induced deficits. However, E12 grafts resulted in cell yields greater than previously reported for untreated primary tissue, with mean TH-positive cell counts in excess of 25,000 neurons, compared with E14 TH cell counts of 4000-5000 cells, representing survival rates of 75% and 12.5%, respectively, based on the expected adult complement. The equivalence of graft induced behavioral recovery between the two graft groups is attributed to a threshold number of cells, above which no further improvement is seen. Such high dopamine cell survival rates should mean that multiple, functioning grafts can be derived from a single embryonic donor, and if similar yields could be obtained from human tissues then the goal of one embryo per patient would be achieved.

Improved survival of young donor age dopamine grafts in a rat model of Parkinson's disease.

In an attempt to improve the survival of implanted dopamine cells, we have readdressed the optimal embryonic donor age for dopamine grafts. In a rat model of Parkinson's disease, animals with unilateral 6-hydroxydopamine lesions of the median forebrain bundle received dopamine-rich ventral mesencephalic grafts derived from embryos of crown to rump length 4, 6, 9, or 10.5 mm (estimated embryonic age (E) 11, E12, E13 and E14 days post-coitus, respectively). Grafts derived from 4 mm embryos survived poorly, with less than 1% of the implanted dopamine cells surviving. Grafts derived from 9 mm and 10.5 mm embryos were similar to those seen in previous experiments with survival rates of 8% and 7% respectively. The best survival was seen in the group that received 6 mm grafts, which were significantly larger than all other graft groups. Mean dopamine cell survival in the 6 mm group (E12) was 36%, an extremely high survival rate for primary, untreated ventral mesencephalic grafts applied as a single placement, and more than fivefold larger than the survival rate observed in the 10.5 mm (E14) group. As E12 ventral mesencephalic tissues contain few, if any, differentiated dopamine cells we conclude that the large numbers of dopamine cells seen in the 6 mm grafts must have differentiated post-implantation. We consider the in vivo conditions which allow this differentiation to occur, and the implications for the future of clinical trials based on dopamine cell replacement therapy.

Delivery of sonic hedgehog or glial derived neurotrophic factor to dopamine-rich grafts in a rat model of Parkinson's disease using adenoviral vectors Increased yield of dopamine cells is dependent on embryonic donor age.

The poor survival of dopamine grafts in Parkinson's disease is one of the main obstacles to the widespread application of this therapy. One hypothesis is that implanted neurons, once removed from the embryonic environment, lack the differentiation factors needed to develop the dopaminergic phenotype. In an effort to improve the numbers of dopamine neurons surviving in the grafts, we have investigated the potential of adenoviral vectors to deliver the differentiation factor sonic hedgehog or the glial cell line-derived neurotrophic factor GDNF to dopamine-rich grafts in a rat model of Parkinson's disease. Adenoviral vectors containing sonic hedgehog, GDNF, or the marker gene LacZ were injected into the dopamine depleted striatum of hemiparkinsonian rats. Two weeks later, ventral mesencephalic cell suspensions were prepared from embryos of donor ages E12, E13, E14 or E15 and implanted into the vector-transduced striatum. Pre-treatment with the sonic hedgehog vector produced a three-fold increase in the numbers of tyrosine hydroxylase-positive (presumed dopaminergic) cells in grafts derived from E12 donors, but had no effect on E13-E15 grafts. By contrast, pre-treatment with the GDNF vector increased yields of dopamine cells in grafts derived from E14 and E15 donors but had no effect on grafts from younger donors. The results indicate that provision of both trophic and differentiation factors can enhance the yields of dopamine neurons in ventral mesencephalic grafts, but that the two factors differ in the age and stage of embryonic development at which they have maximal effects.

In vivo transgene expression from an adenoviral vector is altered following a 6-OHDA lesion of the dopamine system.

We have investigated the in vivo dynamics of an adenovirus-based, LacZ expressing vector, RAd36, at different doses, when injected unilaterally into the corpus striatum of normal rats. We have further investigated the characteristics of this vector in the presence of a 6-OHDA lesion of the nigrostriatal pathway. The dopamine-depleting lesion had an effect on both the number and the distribution of cells transduced by the adenoviral vector. The lesioned side of the brain contained significantly greater numbers of beta-galactosidase positive cells than the unlesioned side at 3 days, 1 week and 4 weeks post-injection and the distribution of transduced cells was altered by the presence of a dopamine lesion. We conclude that the increased levels of transgene expression seen in the lesioned hemisphere are due to a change in the diffusion characteristics of the injected vector in the lesioned hemisphere. These results indicate that, when investigating the use of virus-based vectors, ultimately for use in gene therapies in the CNS, the in vivo dynamics of the vector need to be assessed not only in the normal brain, but also in the pathological brain state such as animal models of target diseases.

Latency associated promoter transgene expression in the central nervous system after stereotaxic delivery of replication-defective HSV-1-based vectors.

The herpes simplex virus type 1 (HSV-1) latency associated promoter (LAP) has been shown to sustain long-term reporter gene expression within sensory neurones. Its activity within the CNS is, however, less well understood. In this study we characterise the activity of the LAP after stereotaxic delivery of recombinant HSV-1-based vectors to the brain. Two classes of vectors were utilised in these studies: (1) a replication-defective vector lacking the glycoprotein H and thymidine kinase genes, designated CS1, and (2) a virus mutant severely impaired for immediate-early (IE) gene expression which lacks functional VP16, ICP4 and ICP0 genes, designated in1388. Both vectors contain the LacZ gene under the control of the LAP. Following delivery of either vector to the striatum, beta-gal expression was detected within anatomically related CNS regions distal to the site of injection. At these sites the number of beta-gal-positive cells increased with time and remained stable up to 4 weeks p.i. beta-Gal expression could not be detected at the site of injection after delivery of CS1 but beta-gal expression within neurones located at this site was observed after delivery of in1388, indicating reduced toxicity of this severely disabled virus. Transgene expression decreased dramatically with both vectors at later time-points (>4 weeks after delivery), but PCR analysis demonstrated that viral genomes were stably maintained for up to 180 days following delivery, indicating that the loss of beta-gal-positive neurones was not likely to be due to a loss of vector-transduced cells. Moreover, after delivery of an equivalent virus to the rat striatum in situ hybridisation analysis showed a similar decrease in the number of neurones expressing the endogenous LATs with time. These data indicate that although the HSV-1 LAP can drive the expression of foreign genes in a variety of CNS neurones, in these cells there is a slow down-regulation of the viral promoter which eventually results in the loss of detectable transgene expression.

The T cell oncogene Tal2 is necessary for normal development of the mouse brain.

Transcription factors are commonly involved in leukemia by activation through chromosomal translocations and normally function in cell type(s) that differ from that of the tumor. TAL2 is a member of a basic helix-loop-helix gene family specifically involved in T cell leukemogenesis. Null mutations of Tal2 have been made in mice to determine its function during development. Tal2 null mutant mice show no obvious defects of hematopoiesis. During embryogenesis, Tal2 expression is restricted to the developing midbrain, dorsal diencephalon, and rostroventral diencephalic/telencephalic boundary, partly along presumptive developing fiber tracts. The null mutant mice are viable at birth but growth become progressively retarded and they do not survive to reproductive age. Tal2-deficient mice show a distinct dysgenesis of the midbrain tectum. Due to loss of superficial gray and optical layers, the superior colliculus is reduced in size and the inferior colliculus is abnormally rounded and protruding. Death is most likely due to progressive hydrocephalus which appears to be caused by obstruction of the foramen of Monro (the connection between the ventricles of the forebrain). Thus, in addition to its oncogenicity when ectopically expressed, Tal2 normally plays a pivotal role in brain development and without this gene, mice cannot survive to maturity.

Antioxidant strategy to counteract the side effects of antipsychotic therapy: an in vivo study in rats.

The effects of long-term administration of the dopamine D(2) receptor antagonist haloperidol on Parkinsonian symptoms have been shown to persist after cessation of the drug treatment. In order to determine whether the level of tyrosine hydroxylase could be affected by subchronic administration of haloperidol, we examined tyrosine hydroxylase-positive immunoreactive cells in the substantia nigra after blockade of dopaminergic receptors with this antipsychotic. Three weeks of injections with haloperidol (1.5 mg/kg, i.p.) caused a significant decrease in tyrosine hydroxylase-positive cell counts at 24 h (27%), 5 days (21%) and 2 weeks (10%) after the last administration, an effect that was blocked by concurrent administration of the antioxidant, vitamin C. The level of tyrosine hydroxylase returned to baseline after 4 weeks withdrawal, no change being observed at later time-points. Nissl staining demonstrated that no damage to the cell bodies was observed, suggesting that the decrease in tyrosine hydroxylase-positive cells was not due to dopaminergic cell loss. These results demonstrate a depleting action of a short course of haloperidol on nigral tyrosine hydroxylase that outlasts the period of application by 2-4 weeks. Moreover, the current study has shown the effect of the antioxidant vitamin C in protecting haloperidol effects on tyrosine hydroxylase-immunostaining.

Behavioral recovery after transplantation into a rat model of Huntington's disease: dependence on anatomical connectivity and extensive postoperative training.

Rats were trained to perform a conditioned stimulus-response task known to be sensitive to striatal damage, after which they received unilateral excitotoxic striatal lesions. The subsequent implantation of graft tissue into the lesioned striatum was either immediate (9 days) or substantially delayed (70 days). When retested 14 weeks later, all graft and lesion rats were equally impaired initially and biased their responding toward the ipsilateral side. Graft-associated recovery was evident with repeated postoperative testing, but only in rats that had received transplants 9 days postlesion. It is suggested that this training-dependent, graft-associated recovery is mediated specifically by the restored host-graft connections.

The influence of excitotoxic basal ganglia lesions on motor performance in the common marmoset.

Huntington's disease is a genetically inherited neurodegenerative disorder for which currently there is no effective treatment or cure. In order to gauge the potential therapeutic benefits of neuroprotective or restorative treatments, it is necessary to create an animal model that is associated with readily measurable and long-lasting functional impairments. The undifferentiated neostriatum and limited behavioural repertoire of rodents have led to the extension of our investigations into the common marmoset. We have used quinolinic acid to create unilateral excitotoxic lesions of the caudate nucleus or the putamen in this small non-human primate. Following rigorous investigation of each monkey on a battery of behavioural tests, we found that the unilateral putamen lesion was associated with a contralateral motor impairment that persisted for at least 9 months and withstood repeated testing. However, the unilateral caudate nucleus lesion did not appear to be associated with any detectable motor deficit. The stability and the reproducibility of the unilateral putamen lesion in the marmoset provide a suitable tool for the investigation of potential treatments for neurodegenerative disorders that attack this region of the brain.

Myelination and behaviour of tenascin-C null transgenic mice.

The extracellular matrix glycoprotein tenascin-C is widely expressed during development and repair, making it surprising that few abnormalities have been found in transgenic mice lacking this molecule. We have therefore re-examined the transgenic mice described by Saga et al. [Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. & Aizawa, S. (1992) Genes Dev., 6 1821-1831] in which tenascin-C was knocked-out by homologous recombination, focusing on two aspects of the nervous system likely to reveal any abnormalities that might follow the loss of tenascin-C. First, we have determined the pattern of myelin and distribution of oligodendrocyte precursor cells in those areas, such as the optic nerve and retina where local concentrations of tenascin-C have been proposed to act as barriers to oligodendrocyte precursor migration and so prevent inappropriate myelination. Secondly, we have examined the behaviour of the mice in a number of well-characterized tests, e.g. beam-walking, passive avoidance and the Morris water maze. We find no abnormalities of myelination or oligodendrocyte precursor distribution in adult mice, showing that local concentrations of tenascin-C are not the sole mechanism responsible for the pattern of myelination in these regions of CNS. However, we do find a number of behavioural abnormalities in these mice and show that hyperlocomotion and deficits in coordination during beam walking can be ascribed to tenascin-C deficiency. The effects on coordination are, however, not seen on a 129 genetic background. Taken together, these results significantly extend the phenotype associated with tenascin-C deficiency but argue against a role in myelination.

Behavioural recovery following striatal transplantation: effects of postoperative training and P-zone volume.

Rats were trained on an operant task and then received striatal lesions and grafts. Grafts were derived either from whole-ganglionic eminences or restricted to the lateral eminence. When retested 4 months later; graft-associated behavioural recovery was only apparent with extensive retesting. There was no difference in performance between rats that received whole-dissection or lateral-dissection grafts, and no correlation between performance and the amount of striatal-like (P-zone) tissue within the graft. It is suggested that P-zone reconstruction may be necessary, but not sufficient for behavioural recovery, which may additionally depend upon rehabilitative training.