In situ endoscopic analysis of bone microstructure and vascularization in post-extraction sites immediately after a minimally invasive vertical tooth extraction in teeth with different periodontal status / Análisis endoscópico in situ de la microestructura ósea y vascularización en alvéolos post-extracción inmediatamente después de una extracción dental mínimamente invasiva en dientes con diferente estado periodontal

SUMMARY: The aim of this study was to perform an in situ endoscopic analysis of the vascularization of post-extraction sites immediately after a non-traumatic extraction in terms of the number of blood vessels per field (NBV), relative area of blood vessels (RABV) and relative area of unmineralized bone (RAUB) in teeth with different periodontal status (PS). This assessment was performed using short distance support immersion endoscopy (SD-SIE). Ten patients (4 men/ 6 women, aged between 25 and 44) were selected. From them, 10 teeth were extracted due to periodontal reasons or other motives. These teeth were then categorized into 2 groups according to their PS, either as periodontally compromised (PC) (clinical attachment loss (CAL) > 7 mm and probing depth (PD) > 5 mm) or periodontally healthy (PH) (CAL < 7 mm and PD < 5 mm, without bleeding or suppuration during periodontal probing), and mobile (M) (> 1 mm horizontally) or immobile (I) (< 1 mm horizontally). The minimally invasive vertical tooth extractions were performed using the Benex ® extractor. Immediately after extraction, a rigid immersion endoscope with a diameter of 2.7 mm was introduced, and a video-alveoloscopy was carried out. This video was analyzed by ImageJ software for the quantification of NBV, RABV and RAUB per field of the post-extraction sites with different PS (PC, PH, M, I) were quantified. In the PC group, significantly greater values for RAUB were observed (33.45 %) compared to those from the PH group (19.65 %). Compared with the M group, the I group did not show significant differences in terms of RAUB or RABV. There were also no differences in NBV in both groups (Means: 33.8 vs. 30.5, respectively).

RESUMEN: El objetivo de este estudio fue realizar un análisis endoscópico in situ de la vascularización de los alvéolos post-extracción inmediatamente después de una extracción atraumática en términos de número de vasos sanguíneos por campo de observación (NBV), área relativa de vasos sanguíneos (RABV) y el área relativa de espacios no mineralizados (RAUB) en dientes con diferente estado periodontal (PS). Esta evaluación se realizó mediante endoscopía de inmersión de corta distancia (SD-SIE). Se seleccionaron diez pacientes (4 hombres / 6 mujeres, con edades comprendidas entre 25 y 44). De ellos, se extrajeron 10 dientes debido a razones periodontales u otros motivos. Estos dientes se clasificaron en 2 grupos según su PS, ya sea como periodontalmente comprometidos (PC), los que presentaban un nivel de inserción clínica (CAL) ≥ 7 mm y una profundidad de sondaje (PD) ≥ 5 mm; o periodontalmente sanos (PH) (CAL <7 mm y PD <5 mm, sin sangramiento o supuración durante el sondaje periodontal). También se categorizaron según su movilidad como móvil (M) (≥ 1 mm horizontalmente) o inmóvil (I) (<1 mm horizontalmente). Las extracciones verticales mínimamente invasivas se realizaron con el extractor Benex ®. Inmediatamente después de la extracción, se introdujo un endoscopio rígido de inmersión con un diámetro de 2.7 mm, con el cual se realizó una video-alveoloscopía. Este video fue analizado por el software ImageJ para la cuantificación de NBV, RABV y RAUB por campo, de los alvéolos post-extracción con diferente estado periodontal. En el grupo de dientes PC, se observaron valores significativamente mayores para RAUB (33.45%) en comparación con los del grupo PH (19.65 %). En comparación con el grupo M, el grupo I no mostró diferencias significativas en términos de RAUB o RABV. Tampoco hubo diferencias en el NBV en ambos grupos (Media: 33.8 frente a 30.5, respectivamente).

In Situ Endoscopic Analysis of Vascular Supply and Regenerated Alveolar Bone in ß-TCP Grafted and Ungrafted Postextraction Sites before Implant Placement: A Prospective Case Control Study.

BACKGROUND: Endoscopy has seen a significant development over recent years in various medical fields with its application expanding from the support of minimal invasive surgery to in situ imaging. In this context, the application of endoscopic techniques to assess the quality of the regenerated bone in situ in the drill hole before implant placement is an appealing approach. AIM: The aim of this study was to use short distance support immersion endoscopy (SD-SIE) to compare the quality of regenerated bone in healed postextraction sites, which are grafted with an in situ hardening ß-TCP, against ungrafted sites, before implant placement. This assessment was based on microscopic bone analysis in combination with the blood vessel count. METHOD: 13 spontaneously healed and 13 grafted postextraction sites in 3 men and 6 women, aged 26-83 years, were evaluated using SD-SIE after 4-6 months. SD-SIE was applied in drill holes before implant placement, and videos were taken from representative central buccal areas. The video recordings were analyzed using Image J software for (1) number of blood vessels per area (NBV), (2) relative area of vessels (VA), (3) relative area of mineralized bone (MBA), (4) relative area of unmineralized bone (UMBA), and (5) relative area of bone substitute (BSA). RESULTS: The grafted sites showed more (1) NBV as well as (2) VA (8.6 ± 1.1; 2.03 ± 0.28%) than the ungrafted sites (2.5 ± 0.6; 1.18 ± 0.36%) (independent t-test; p < 0.05); (3) MBA and (4) UMBA were similar to those in the grafted sites (86.3 ± 2.2 %; 13.7 ± 2.2 %) and to the ungrafted sites (89.5 ± 3.7%; 10.5 ± 3.6%) (independent t-test; p > 0.05); and (5) BSA in the grafted sites was 18.2 ± 5.4%. CONCLUSION: SD-SIE is an interesting new approach for in situ assessment of bone quality and blood supply before implant placement. The regenerated bone in ß-TCP grafted extraction sockets showed an increased vascularization compared to ungrafted sites providing a vital support for subsequent implant placement.

Hard Tissue Preservation in Minimally Invasive Mandibular Third Molar Surgery Using In Situ Hardening TCP Bone Filler.

BACKGROUND: Maintenance of hard tissue in the case of impacted third molars (M3M) with close relationship to the mandibular canal is still a surgical challenge which may be overcome using the inward fragmentation technique. METHODS: A consecutive case series of 12 patients required the extraction of 13 impacted M3M with a close relationship to the inferior alveolar nerve (IAN). Via occlusal miniflaps, M3M were exposed occlusal under endoscopic vision and removed by inward fragmentation. All patients received socket preservation with resorbable in situ hardening TCP particles to reduce the risk of pocket formation at the second molar. RESULTS: All 13 sites healed uneventfully. Bone height was assessed using CBCT cross-sectional reformats pre- and 3 months postoperatively. The bone height was reduced by 1.54 mm lingual (SD 0.88), 2.91 mm central (SD 0.93), and 2.08 mm buccal (SD 1.09). Differences were significant at a 0.05% level. No tissue invagination at the extraction sites was observed. CONCLUSIONS: Major bone defects can be avoided safely using inward fragmentation surgery. The self-hardening bone filler appears to enhance the mineralization of the intrabony defect.

µCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.

The small size of the adult and developing mouse heart poses a great challenge for imaging in preclinical research. The aim of the study was to establish a phosphotungstic acid (PTA) ex-vivo staining approach that efficiently enhances the x-ray attenuation of soft-tissue to allow high resolution 3D visualization of mouse hearts by synchrotron radiation based µCT (SRµCT) and classical µCT. We demonstrate that SRµCT of PTA stained mouse hearts ex-vivo allows imaging of the cardiac atrium, ventricles, myocardium especially its fibre structure and vessel walls in great detail and furthermore enables the depiction of growth and anatomical changes during distinct developmental stages of hearts in mouse embryos. Our x-ray based virtual histology approach is not limited to SRµCT as it does not require monochromatic and/or coherent x-ray sources and even more importantly can be combined with conventional histological procedures. Furthermore, it permits volumetric measurements as we show for the assessment of the plaque volumes in the aortic valve region of mice from an ApoE-/- mouse model. Subsequent, Masson-Goldner trichrome staining of paraffin sections of PTA stained samples revealed intact collagen and muscle fibres and positive staining of CD31 on endothelial cells by immunohistochemistry illustrates that our approach does not prevent immunochemistry analysis. The feasibility to scan hearts already embedded in paraffin ensured a 100% correlation between virtual cut sections of the CT data sets and histological heart sections of the same sample and may allow in future guiding the cutting process to specific regions of interest. In summary, since our CT based virtual histology approach is a powerful tool for the 3D depiction of morphological alterations in hearts and embryos in high resolution and can be combined with classical histological analysis it may be used in preclinical research to unravel structural alterations of various heart diseases.

SK3 Channel Overexpression in Mice Causes Hippocampal Shrinkage Associated with Cognitive Impairments.

The dysfunction of the small-conductance calcium-activated K+ channel SK3 has been described as one of the factors responsible for the progress of psychoneurological diseases, but the molecular basis of this is largely unknown. This report reveals through use of immunohistochemistry and computational tomography that long-term increased expression of the SK3 small-conductance calcium-activated potassium channel (SK3-T/T) in mice induces a notable bilateral reduction of the hippocampal area (more than 50 %). Histological analysis showed that SK3-T/T mice have cellular disarrangements and neuron discontinuities in the hippocampal formation CA1 and CA3 neuronal layer. SK3 overexpression resulted in cognitive loss as determined by the object recognition test. Electrophysiological examination of hippocampal slices revealed that SK3 channel overexpression induced deficiency of long-term potentiation in hippocampal microcircuits. In association with these results, there were changes at the mRNA levels of some genes involved in Alzheimer's disease and/or linked to schizophrenia, epilepsy, and autism. Taken together, these features suggest that augmenting the function of SK3 ion channel in mice may present a unique opportunity to investigate the neural basis of central nervous system dysfunctions associated with schizophrenia, Alzheimer's disease, or other neuropsychiatric/neurodegenerative disorders in this model system. As a more detailed understanding of the role of the SK3 channel in brain disorders is limited by the lack of specific SK3 antagonists and agonists, the results observed in this study are of significant interest; they suggest a new approach for the development of neuroprotective strategies in neuropsychiatric/neurodegenerative diseases with SK3 representing a potential drug target.

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1(+/+), CX3CR1(+/GFP) and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1(+/GFP) mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.

Support Immersion Endoscopy in Post-Extraction Alveolar Bone Chambers: A New Window for Microscopic Bone Imaging In Vivo.

Using an endoscopic approach, small intraoral bone chambers, which are routinely obtained during tooth extraction and implantation, provide visual in vivo access to internal bone structures. The aim of the present paper is to present a new method to quantify bone microstructure and vascularisation in vivo. Ten extraction sockets and 6 implant sites in 14 patients (6 men / 8 women) were examined by support immersion endoscopy (SIE). After tooth extraction or implant site preparation, microscopic bone analysis (MBA) was performed using short distance SIE video sequences of representative bone areas for off-line analysis with ImageJ. Quantitative assessment of the microstructure and vascularisation of the bone in dental extraction and implant sites in vivo was performed using ImageJ. MBA revealed bone morphology details such as unmineralised and mineralised areas, vascular canals and the presence of bleeding through vascular canals. Morphometric examination revealed that there was more unmineralised bone and less vascular canal area in the implant sites than in the extraction sockets.

Doxycycline restrains glia and confers neuroprotection in a 6-OHDA Parkinson model.

Neuron-glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6-OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6-OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6-OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.

Role of nitric oxide in motor control: implications for Parkinson's disease pathophysiology and treatment.

According to classical thinking about Parkinson's disease, loss of dopaminergic input from the substantia nigra pars compacta leads to overactivity and underactivity of the indirect and direct output pathways, respectively, in the basal ganglia. Administration of the dopamine precursor L-DOPA (l-3, 4-dihydroxyphenylalanine) is proposed to induce changes in the opposite directions. L-DOPA is the most used drug to treat Parkinson`s disease symptoms. After repeated treatment with this compound, however, disabling secondary effects such as the abnormal involuntary movements usually appear. Nitric oxide is a free radical that can also act as an atypical neurotransmitter and influences dopamine-mediated neurotransmission. In this paper we will briefly review the role of nitric oxide on motor control and in Parkinson's disease, particularly a possible role of nitric oxide in L-DOPA induced dyskinesia in rodents. Recent results show that nitric oxide synthase inhibition reduces L-DOPA-induced dyskinesia in rats and mice. The effect is dose-dependent, does not suffer tolerance nor interferes with L-DOPA positive motor effects. These preclinical findings suggest that nitric oxide is a promising therapeutic target for the reduction of L-DOPA-induced dyskinesia.

Effect of Tityus serrulatus scorpion venom and its major toxin, TsTX-I, on the complement system in vivo.

The effects of Tityus serrulatus venom and TsTX-I (Ts1 or gamma-toxin) on the lytic activity of the complement system (CS) were investigated in vivo. Serum classical pathway (CP) and alternative pathway (AP) activities were determined in sera of rats (200+/-10 g) injected i.p. with soluble venom (150 microg/kg), TsTX-I (150 microg/kg) or saline (control). The animals were sacrificed 0.5, 1, 2, 4, 24 and 48 h after injection. The results showed an increase in serum lytic activity of animals injected with venom, reaching values up to 70% above controls in CP activity and 120% in AP activity. These effects were biphasic with maximum values 1 and 24 h after venom injection. Similar effects were obtained for TsTX-I, but with lower intensity. Hematocrit values of all tested animals were determined to evaluate the effect of hemoconcentration on the lytic activity of the CS. It was observed that the maximum hematocrit value was obtained 1 h after injection and returned to normal values within 24 h. These data indicate that hemoconcentration can play a relevant role in the first peak of complement activity, but we cannot discard a direct action of the venom on the system during this period, since the serum venom concentration is maximal 15-30 min after envenomation. The high lytic activity of the serum observed after 24 h, period in which the hematocrit values are normal and no venom can be detected, may be consequence of the inflammatory process induced by the venom or toxin. The lytic activity of the serum of rats injected with venom, TsTX-I or saline was abolished when the serum was previously adsorbed on zymosan. These data confirm that the increase of the lytic activity of the serum induced by the venom or toxin is dependent on CS. These results show that CS is involved in the inflammatory process induced by the venom or toxin and consequently in the lung edema, hemolysis, leukocytosis, among other clinical manifestations of severe envenomation.