Biofilm and cell adhesion strength on dental implant surfaces via the laser spallation technique.

OBJECTIVE: The aims of this study are to quantify the adhesion strength differential between an oral bacterial biofilm and an osteoblast-like cell monolayer to a dental implant-simulant surface and develop a metric that quantifies the biocompatible effect of implant surfaces on bacterial and cell adhesion. METHODS: High-amplitude short-duration stress waves generated by laser pulse absorption are used to spall bacteria and cells from titanium substrates. By carefully controlling laser fluence and calibration of laser fluence with applied stress, the adhesion difference between Streptococcus mutans biofilms and MG 63 osteoblast-like cell monolayers on smooth and rough titanium substrates is obtained. The ratio of cell adhesion strength to biofilm adhesion strength (i.e., Adhesion Index) is determined as a nondimensionalized parameter for biocompatibility assessment. RESULTS: Adhesion strength of 143 MPa, with a 95% C.I. (114, 176), is measured for MG 63 cells on smooth titanium and 292 MPa, with a 95% C.I. (267, 306), on roughened titanium. Adhesion strength for S. mutans on smooth titanium is 320 MPa, with a 95% C.I. (304, 333), and remained relatively constant at 332 MPa, with a 95% C.I. (324, 343), on roughened titanium. The calculated Adhesion Index for smooth titanium is 0.451, with a 95% C.I. (0.267, 0.622), which increased to 0.876, with a 95% C.I. (0.780, 0.932), on roughened titanium. SIGNIFICANCE: The laser spallation technique provides a platform to examine the tradeoffs of adhesion modulators on both biofilm and cell adhesion. This tradeoff is characterized by the Adhesion Index, which is proposed to aid biocompatibility screening and could help improve implantation outcomes. The Adhesion Index is implemented to determine surface factors that promote favorable adhesion of cells greater than biofilms. Here, an Adhesion Index ≫ 1 suggests favorable biocompatibility.

Adhesion of biofilms on titanium measured by laser-induced spallation.

Eradication of established implant-associated and bacterial biofilm-forming infections remains difficult in part because these biofilms remain well-adhered to the implant surface. Few experimental techniques are available to measure macro-scale strength of bacterial biofilm-implant adhesion. We have adapted the laser spallation technique to compare the macro-scale adhesion strength of biofilms formed on titanium. By using a rapid pressure wave (35 ns) to load the interface, we prevent disturbance of the biofilm surface prior to measurement, and preclude the time necessary for the biofilm to respond to and adapt under loading. Biofilms of Streptococcus mutans, a Gram-positive bacterium associated with human dental caries (cavities) were cultured directly on commercially pure titanium within our custom substrate assembly. Growth conditions were varied by adding sucrose to the Todd Hewitt Yeast (THY) broth: THY control, 37.5 mM, 75 mM, 375 mM, and 750 mM sucrose. Multiple locations on each biofilm were loaded using the laser spallation technique. Loading pressure wave amplitude was controlled by adjusting laser fluence, energy per area. Initially, addition of sucrose to the media increased biofilm adhesion to titanium. However, once a saturation concentration of 75 mM sucrose was reached, increasing the sucrose concentration further resulted in a decrease in biofilm adhesion. This study is the first demonstration of the adaptation of the laser spallation technique to measure bacterial biofilm adhesion. Establishment of this macro-scale biofilm adhesion measurement technique opens the door for many biofilm-surface adhesion studies. We anticipate further work in this area towards understanding the complex relationships among bacteria species, environmental factors, surface characteristics, and biofilm adhesion strength.

Isoflurane affects the cytoskeleton but not survival, proliferation, or synaptogenic properties of rat astrocytes in vitro.

BACKGROUND: More than half of the cells in the brain are glia and yet the impact of general anaesthetics on these cells is largely unexamined. We hypothesized that astroglia, which are strongly implicated in neuronal well-being and synapse formation and function, are vulnerable to adverse effects of isoflurane. METHODS: Cultured rat astrocytes were treated with 1.4% isoflurane in air or air alone for 4 h. Viability, proliferation, and cytoskeleton were assessed by colorimetric assay, immunocytochemistry, or a migration assay at the end of treatment or 2 days later. Also, primary rat cortical neurones were treated for 4 days with conditioned medium from control [astrocyte-conditioned media (ACM)], or isoflurane-exposed astrocytes (Iso-ACM) and synaptic puncta were assessed by synapsin 1 and PSD-95 immunostaining. RESULTS: By several measures, isoflurane did not kill astrocytes. Nor, based on incorporation of a thymidine analogue, did it inhibit proliferation. Isoflurane had no effect on F-actin but reduced expression of α-tubulin and glial fibrillary acidic protein both during exposure (P<0.05 and P<0.001, respectively) and 2 days later (P<0.01), but did not impair astrocyte motility. ACM increased formation of PSD-95 but not synapsin 1 positive puncta in neuronal cultures, and Iso-ACM was equally effective. CONCLUSIONS: Isoflurane decreased expression of microtubule and intermediate filament proteins in astrocytes in vitro, but did not affect their viability, proliferation, motility, and ability to support synapses.

Effective release rates at single rat Schaffer collateral-CA1 synapses during sustained theta-burst activity revealed by optical imaging.

To understand how information is coded at single hippocampal synapses during high-frequency activity, we imaged NMDA receptor-mediated Ca(2+) responses in spines of CA1 neurons using two-photon microscopy. Although discrete quantal events were not readily apparent during continuous theta-burst stimulation (TBS), we found that the steady-state dendritic Ca(2+) response was spatially restricted (half-width < 1 microm), voltage dependent and sensitive to MK-801, indicating that that it was mediated by activation of NMDA receptors at single synapses. Partial antagonism of NMDA receptors caused a similar reduction of NMDA EPSCs (measured at the soma) and local dendritic Ca(2+) signals, suggesting that, like EPSCs, the steady-state Ca(2+) signal was made up of a linear addition of quantal events. Statistical analyses of the steady-response suggested that the quantal size did not change dramatically during TBS. Deconvolution of TBS-evoked Ca(2+) responses revealed a heterogeneous population of synapses differing in their capacity to signal high-frequency information, with an average effective steady-state release rate of approximately 2.6 vesicles synapse(-1)s(-1). To assess how the optically determined release rates compare with population measures we analysed the rate of decay of peak EPSCs during train stimulation. From these studies, we estimated a unitary vesicular replenishment rate of 0.02 s(-1), which corresponds to an average release rate of approximately 0.8-2 vesicles s(-1) at individual synapses. Additionally, extracellular recordings from single Schaffer collaterals revealed that spikes propagate reliably during TBS. Hence, during high-frequency activity, Schaffer collaterals conduct spikes with high fidelity, but release quanta with relatively lower efficiency, leaving NMDA receptor function largely intact and synapse specific. Heterogeneity in release rates between synapses suggests that similar patterns of presynaptic action potentials could trigger different forms of plasticity at individual synapses.

A comparison of koniocellular, magnocellular and parvocellular receptive field properties in the lateral geniculate nucleus of the owl monkey (Aotus trivirgatus).

1. By analogy to previous work on lateral geniculate nucleus (LGN) magnocellular (M) and parvocellular (P) cells our goal was to construct a physiological profile of koniocellular (K) cells that might be linked to particular visual perceptual attributes. 2. Extracellular recordings were used to study LGN cells, or their axons, in silenced primary visual cortex (V1), in nine anaesthetized owl monkeys injected with a neuromuscular blocker. Receptive field centre-surround organization was examined using flashing spots. Spatial and temporal tuning and contrast responses were examined using drifting sine-wave gratings; counterphase sine-wave gratings were used to examine linearity of spatial summation. 3. Receptive fields of 133 LGN cells and 10 LGN afferent axons were analysed at eccentricities ranging from 2.8 to 31.3 deg. Thirty-four per cent of K cells and only 9 % of P and 6 % of M cells responded poorly to drifting gratings. K, P and M cells showed increases in centre size with eccentricity, but K cells showed more scatter. All cells, except one M cell, showed linearity in spatial summation. 4. At matched eccentricities, K cells exhibited lower spatial and intermediate temporal resolution compared with P and M cells. K contrast thresholds and gains were more similar to those of M than P cells. M cells showed lower spatial and higher temporal resolution and contrast gains than P cells. 5. K cells in different K LGN layers differed in spatial, temporal and contrast characteristics, with K3 cells having higher spatial resolution and lower temporal resolution than K1/K2 cells. 6. Taken together with previous results these findings suggest that the K cells consist of several classes, some of which could contribute to conventional aspects of spatial and temporal resolution.

Organization of efferent neurons in area 19: the projection to extrastriate area 21a.

The organization of efferent neurons in area 19 of the cat was examined by bulk injections of retrograde tracers, WGA-HRP and CTX-Au, into extrastriate area 21a. In one case, the cortex was cut coronally and retrogradely labeled cells in area 19 were present in columnar register throughout layers 2 to 6, with the majority of labeled cells in layers 2/3. The number of columns per tissue section ranged from 0 to 4 and had a centre-to-centre spacing ranging from 0.6 to 0.9 mm. A few lightly labeled cells were found between the columns. In six other cases, the visual cortex was flattened, and cut in the tangential plane to reveal a pattern of irregular, widely spaced bands that were elongated in the mediolateral direction with a mean centre-to-centre spacing of 2.6 mm. The density of labeled cells within these bands fluctuated such that dense aggregates of cells were found, on average, at 0.9 mm intervals along the bands. This tangential heterogeneity in density, along with the patchy columnar staining witnessed in the coronal plane, suggests that the widely spaced efferent projection bands may have a patchy substructure with a spacing of approximately 1 mm. The pattern of efferent projection bands and its substructure in area 19 is reminiscent of the stripe-like organization of V2 found in primates.

The connections of layer 4 subdivisions in the primary visual cortex (V1) of the owl monkey.

The primary visual cortex (V1) of primates receives signals from parallel lateral geniculate nucleus (LGN) channels. These signals are utilized by the laminar and compartmental [i.e. cytochrome oxidase (CO) blob and interblob] circuitry of V1 to synthesize new output pathways appropriate for the next steps of analysis. Within this framework, this study had two objectives: (i) to analyze the con- nections between primary input and output layers and compartments of V1; and (ii) to determine differences in connection patterns that might be related to species differences in physiological properties in an effort to link specific pathways to visual functions. In this study we examined the intrinsic interlaminar connections of V1 in the owl monkey, a nocturnal New World monkey, with a special emphasis on the projections from layer 4 to layer 3. Interlaminar connections were labeled via small iontophoretic or pressure injections of tracers [horseradish peroxidase, biocytin, biotinylated dextrine amine (BDA) or cholera toxin subunit B conjugated to colloidal gold particles]. Our most significant finding was that layer 4 (4C of Brodmann) can be divided into three tiers based upon projections to the superficial layers. Specifically, we find that 4alpha (4Calpha), 4beta (4Cbeta) and 4ctr send primary projections to layers 3C (4B), 3Bbeta (4A) and 3Balpha (3B), respectively. Examination of laminar structure with Nissl staining supports a tripartite organization of layer 4. The cortical output layer above layer 3Balpha (3B) (e.g. layer 3A) does not appear to receive any direct connections from layer 4 but receives heavy input from layers 3Balpha (3B) and 3C (4B). Some connectional differences also were observed between the subdivisions of layer 3 and the infragranular layers. No consistent differences in connections were observed that distinguished CO blobs from interblobs or that could be correlated with differences in visual lifestyle (nocturnal versus diurnal) when compared with connectional data in other primates. Re-examination of data from previous studies in squirrel and macaque monkeys suggests that the tripartite organization of layer 4 and the unique projection pattern of layer 4ctr are not restricted to owl monkeys, but are common to a number of primate species.

The projection from V1 to extrastriate area 21a: a second patchy efferent pathway colocalizes with the CO blob columns in cat visual cortex.

The different patchy organizations of neurons projecting from primary visual cortex (area 17) to the various extrastriate areas may contribute to functional differences in the output to each of these areas. The pattern of neurons projecting to extrastriate area 21a was examined using large injections of retrograde tracers and compared to the pattern shown by neurons projecting to the lateral suprasylvian area (LS). Patches of neurons projecting to 21a showed a bimodal laminar distribution, with numerous labeled cells in the upper and lower third of layer 3 bracketing a sparsely labeled central third; LS-projecting neurons were confined to the lower and middle thirds of layer 3. The 21a projecting cells were relatively tighter in their clustering pattern than the LS projecting cells, i. e. the difference in labeling density between patch and interpatch zones was greater for 21a-projecting cells than for LS-projecting cells. As previously shown for the LS-projecting cells, patches of 21a-projecting cells colocalized with CO blob columns in area 17. Combined with our earlier results, this study shows that the CO blob compartments in area 17 give rise to at least two distinct efferent pathways, one projecting to LS and the other to 21a, and furthermore suggest that each pathway may carry unique information to its extrastriate target.

Projections from V1 to lateral suprasylvian cortex: an efferent pathway in the cat's visual cortex that originates preferentially from CO blob columns.

The patchy pattern of retrograde labeling produced by injections of anatomical tracers into the lateral suprasylvian (LS) visual area was compared to the cytochrome oxidase (CO) blobs in cat visual cortex. Following large injections of anatomical tracers in LS, retrograde labeling formed an irregular lattice of patches with a spacing of slightly less than 1 mm in area 17, and slightly greater than 1 mm in area 18. By comparing labeling in alternate serial sections, patches of LS-projecting cells in both areas were found to align with CO blobs. The conclusion of alignment between CO blob columns and patches of LS-projecting cells was confirmed by a quantitative analysis which showed a significant correlation between the local density of LS-projecting cells in reconstructions of charted cells and the intensity of CO staining in the CO-reacted sections. As for areas 17 and 18, labeling in other afferent areas of LS was also patchy with a spacing on the order of 1 mm except for area 19 where we found patches of LS-projecting cells with a larger spacing, roughly 2 mm. No matching fluctuations in CO density could be discerned in area 19, however. In conjunction with recent evidence that CO blob columns in cats receive strong input from Y-cells of the lateral geniculate nucleus (Boyd & Matsubara, 1996; Shoham, et al., 1996), these data support the hypothesis (Shipp & Grant, 1991) that the patches of LS-projecting cells correspond to Y-cell input columns. As a relationship between the CO architecture and certain classes of efferent cells has previously been shown in primates, these findings show new similarities between CO blobs in different mammalian species.

Relationships between cytochrome oxidase (CO) blobs in primate primary visual cortex (V1) and the distribution of neurons projecting to the middle temporal area (MT).

The cytochrome oxidase (CO) blobs and interblobs in layer 3B of primate visual cortex have different sets of corticocortical connections. Cortical layers below layer 3B also project corticocortically, but the relationship of efferent projections from the deeper layers to the overlying blob/interblob architecture is less clear. We studied the tangential organization of neurons projecting from primary visual cortex (V1) to the middle temporal visual area (MT) and their relationship to the CO blobs. MT-projecting neurons in two primate species, bush babies and owl monkeys, were retrogradely labeled, then charted in tangential sections, and compared to the positions of the overlying CO blobs. In both primate species, MT-projecting neurons in layer 3C were unevenly distributed in the tangential plane, with dense patches of labeled cells that were aligned with the CO blobs. A novel two-dimensional spatial correlation method was used to show the colocalization of MT-projecting cells with the overlying blobs. Chi-square analyses performed with the cortical surface equally divided into compartments of blob, interblob, and blob/interblob borders showed that blob columns tended to have about 1.5 times more MT-projecting cells (P < 0.0001) than interblob columns. Similar analyses were applied to published data on V1 cells projecting to area MT in macaque monkey (Shipp and Zeki [1989] Euro J Neurosci 1:310-332). Again, the results showed a significant correlation between the cell distribution and CO blobs. Taken together, these results suggest that layer 3C is not uniform but is made up of a mosaic of cells that project to area MT and cells that project to some other location. These findings also indicate that the mosaic organization of layer 3C is related in some unique way to the overlying CO architecture.

Addition of droperidol to morphine administered by the patient-controlled analgesia method: what is the optimal dose?

Eighty patients were recruited into a double-blind, randomized trial to find the optimal dose of droperidol for addition to the patient-controlled analgesia (PCA) morphine infusate for female patients undergoing gynaecological surgery. A standardized anaesthetic technique was employed. Post-operative analgesia was provided by PCA morphine. Patients were allocated at random into one of four treatment groups receiving with each PCA morphine bolus: (1) droperidol 0.05 mg; (2) droperidol 0.10 mg; (3) droperidol 0.15 mg; and (4) droperidol 0.20 mg, respectively. The incidence of post-operative nausea and vomiting (PONV), requests for rescue anti-emetic medication, and incidence of side effects were recorded. The number of symptom-free patients in each group increased as the droperidol dose increased, but although there was a significant inverse association between the total dose of droperidol received and the severity of PONV (P < 0.05), there were no significant differences between individual groups. In each group, patients were significantly less sedated at 24 h compared with 12 h (P < 0.01). However, after 24 h, patients in group 4 were significantly more sedated than patients in groups 1 and 2 (P < 0.05). There were no significant differences between the groups in terms of the incidence of anxiety or other side effects attributable to droperidol. The present authors suggest that, although the results demonstrate few statistically significant differences between the four groups, a PCA bolus dose of droperidol of 0.10 mg mL-1 appears to provide the optimal balance between anti-emetic efficacy and an acceptable incidence of side effects.

Nasotracheal intubation in the presence of frontobasal skull fracture.

PURPOSE: To present a case of maxillofacial trauma and basal skull fracture (BSF) in whom nasotracheal intubation (NTI) was successfully used, without complication, to facilitate surgical fixation. To present alternative methods of airway management in this situation and to review the evidence supporting the notion that NTI is contraindicated in the presence of basal skull fracture. CLINICAL FEATURES: A 17-yr-old man was referred for surgical fixation of bilateral mandibular fractures. Cranial computed tomography revealed intracranial air and blood in all four sinuses and distortion of the nasal passage on the right. There was no cerebral injury and the left nasal passage appeared patent. In order to facilitate intraoperative intermaxillary fixation fibreoptic NTI was undertaken in preference to tracheostomy. The patient made an uneventful recovery without evidence of meningitis or direct cerebral injury. CONCLUSION: In selected patients NTI may be performed in the presence of BSF. Available evidence suggests that BSF-should not be regarded as an absolute contraindication to NTI.

Clinical hypnosis for rapid recovery from dissociative identity disorder.

Numerous anecdotal reports and textbooks from distinguished mental health practitioners have accorded to clinical hypnosis a prominent role in the successful psychotherapeutic treatment of Dissociative Identity Disorder (DID). This ideographic inquiry of one such treatment offers empirical support for the instrumental effects of clinical hypnosis with a highly hypnotizable patient who alleged intermittent sexual abuse during the 2-5 age range. Multiple pre- and post-intervention measures, plus treatment trajectory monitoring and explication, demonstrate how hypnosis methodology contributed to a rapid recovery from DID. Future research is called for to further elucidate the therapeutic benefits of clinical hypnosis for DID patients having different levels of hypnotizability and treatment responsiveness.

Laminar and columnar patterns of geniculocortical projections in the cat: relationship to cytochrome oxidase.

We examined the laminar and columnar arrangement of projections from different layers of the lateral geniculate nucleus (LGN) to the visual cortex in the cat. In light of recent reports that cytochrome oxidase blobs (which in primates receive specific geniculate inputs) are also found in the visual cortex of cats, the relationship between cytochrome oxidase staining and geniculate inputs in this species was studied. Injections of wheat germ agglutinin-conjugated horseradish peroxidase were made into the anterior "genu" of the LGN, where isoelevation contours of the geniculate layers are distorted due to the curvature of the nucleus. Consequently, anterograde labeling from the various LGN layers was topographically separated across the surface of the cortex, and labeling in a particular isoelevation representation of the cortex could be associated with a specific layer of the LGN. Labeling from the A layers, which contain X and Y cells, was coextensive with layers 4 and 6 in both area 17 and area 18, as previously reported. Labeling from the C layers, which contain Y and W cells, occupied a zone extending from the 4a/4b border to part way into layer 3 in area 17. The labeling extended throughout layer 4 in area 18. There was also labeling in layer 5a and layer 1 in both area 17 and area 18. Except in layer 1, labeling from the C layers was patchy. In the tangential plane, adjacent sections stained for cytochrome oxidase showed that the patches of labeling from the C laminae aligned with the cytochrome oxidase blobs. The cytochrome blobs were visible in layers 3 and 4a, but not in layer 4b in both areas 17 and 18. These results suggest that W cells project specifically to the layer 3 portion of the blobs, while Y cells, at least those of the C layers, project specifically to the layer 4a portion of the blobs in area 17. The heavy synaptic drive of the Y cells is probably the cause of the elevated metabolism, and thus, higher cytochrome oxidase activity, of the blobs.

Presence of GABA-immunoreactive neurons within intracortical patches in area 18 of the cat.

In cat visual cortex, horizontal, intracortical connections spread laterally to link together specific columnar sites. When visualized by retrograde tracers, these intracortical connections appear as periodic, columnar patches of dense cellular labeling interspersed with areas of much less dense labeling. We looked for anatomical evidence for direct inhibition among the patchy, horizontal connections in area 18, by combining retrograde labeling using wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) with immunohistochemistry using an antiserum against the inhibitory neurotransmitter gamma-amino butyric acid (GABA). We found numerous double-labeled cells associated with some, but not all, of the local patches nearest to the injection site. In the superficial layers, the GABA-immunoreactive cells also labeled with WGA-HRP were confined to a zone approximately 1.0 mm from the center of the injection, while the double-labeled cells in the deeper layers spanned greater distances, up to 3.0 mm from the injection center. These more distant, double-labeled cells in the deeper layers were located on the edges or outside of the patches of dense labeling. Thus, all of the more distant intracortical patches, as well as some of the more proximal patches were devoid of double-labeled cells--a finding which suggests that direct inhibition may occur among only a selected group of the 'short range' intracortical patches and among none of the long-range patches.