Fluorescence immunohistochemistry and confocal scanning laser microscopy

We have developed a reliable technique for labeling and examining neural structures in soft tissues associated with articular joints and have tested it in human wrist joints under various specimen-related conditions. The labeling protocol employs an immunohistochemical process with a panneuronal marker (PGP 9.5) as the primary antibody and Alexa Fluor 488 as the fluorescing secondary antibody. Imaging was done using a confocal laser scanning microscope, which produced exceptionally detailed three-dimensional images of nerve endings and transiting nerve fibers from thick sections of wrist joint ligaments obtained from human cadavers. The protocol provided a practical postmortem window for specimen acquisition and processing without significant apparent worsening of image quality. The images produced are resistant to fading with repeated exposure to a fluorescent light source, which gives many opportunities for observation. Background staining is minimal, producing high contrast labeling of target tissues, which, in turn, enhances image analysis.     

Dural Tissue Trauma and Cerebrospinal Fluid Leak after Epidural Needle Puncture: Effect of Needle Design, Angle, and Bevel Orientation

Background: The effects of epidural needle design, angle, and bevel orientation on cerebrospinal fluid leak after puncture have not been reported. The impact of these factors on leak rate was examined using a dural sac model. Dural trauma was examined using scanning electron microscopy.

Methods: Human cadaveric dura, mounted on a cylindrical model, was punctured with epidural needles using a micromanipulator. Tissue was punctured at 15 cm H2O (left lateral decubitus) system pressure, and leak was measured at 25 cm H2O (semisitting) pressure. Leak rates and trauma were compared for the following: (1) six different epidural needles at 90[degrees], bevel parallel to the dural long axis; (2) 18-gauge Tuohy and 18-gauge Special Sprotte(R) epidural needles, 30[degrees]versus 90[degrees]; (3) 18-gauge Tuohy, bevel perpendicular versus parallel to the dural long axis.

Results: With the 90[degrees] puncture, bevel parallel, the greatest leak occurred with a 17-gauge Hustead (516 +/- 319 ml/15 min), and the smallest leak occurred with a 20-gauge Tuohy (100 +/- 112 ml/15 min; P = 0.0018). A 20-gauge Tuohy puncture led to statistically significant reductions in leak (P value range, 0.0001-0.0024) compared with all needles except the Special Sprotte(R). With the 30[degrees]versus 90[degrees] angle, 30[degrees] punctures with an 18-gauge Tuohy produced nonstatistically significant leak reductions compared with the 18-gauge Tuohy at 90[degrees]. The puncture angle made no difference for the Special Sprotte(R). Nonsignificant reductions were found for the Special Sprotte(R) compared with the Tuohy. With the 18-gauge Tuohy bevel orientation, perpendicular orientation produced nonstatistically significant reductions in leak compared with parallel orientation.     

Reverse pressure softening: a simple tool to prepare areola for easier latching during engorgement.

Successful breastfeeding requires efficient milk transfer through the nipple-areolar complex, which includes subareolar tissue. Subareolar tissue resistance increases during engorgement, when expanded circulation and excess interstitial fluid compete for space with increasing milk volumes. Physiologic and iatrogenic events often combine to produce distortion of breast anatomy. Resulting latch difficulty, delayed milk ejection reflex, poor milk transfer, pain, and nipple damage discourage many mothers. The rationale and technique for a simple intervention developed in practice are described: reverse pressure softening (RPS) before latching significantly reduces resistance of subareolar tissue, temporarily freeing it to interact more efficiently with the baby's mouth. RPS also triggers the milk ejection reflex promptly. The health care provider can perform RPS or teach the mother and her significant others, even by telephone.     

Guided bone regeneration for immediate non‐submerged implant placement using bioabsorbable materials in Beagle dogs

The aim of the present study was to evaluate the combined application of different bioabsorbable materials for healing of residual peri‐implant defects after placement of non‐submerged implants into fresh extraction sockets. Second and third mandibular premolars were extracted from 10 Beagle dogs, the coronal part of the distal sockets were surgically enlarged and this was followed by immediate placement of specially designed hollow‐screw non‐submerged dental implants. For each animal, the coronal peri‐implant defects were further treated with one of the 4 following procedures: 1) no treatment, control site: 2) grafting with porous hydroxyapatite (HA); 3) collagen membrane tightly secured around the implant and over the defect and 4) grafting with HA covered with a collagen membrane. After 16 weeks of healing, specimens were removed from the mandibule and prepared for a histomorphometric evaluation. The bone-to-implant contact length (BIC) was measured and compared amongst the different treatment modalities. In the defect area, the irregular bone regeneration was similar between all the treatment procedures ( P >0.10). In the sites covered with a collagen membrane alone, the total BIC (47%) was greater than in control sites (28.7%. P <0.05) or sites grafted with HA (22.2%, P <0.02). Total BIC in sites treated with the HA‐membrane combination (43%) was only significantly different from sites treated with HA ( P <0.10). It is concluded that the use of bioabsorbable materials results in a limited increase of osseointegration when used in conjunction with immediate placement of non-submerged implants, although the principle of the one stage surgical approach can be maintained.     

Pre-adaptation, adaptation and de-adaptation to high altitude in humans: hormonal and biochemical changes at sea level

High altitude residence is known to modify body biochemistry and hormone status. However, the effects of such a sojourn on these status observed at sea level both immediately and later after return are not as well established as are the effects of an intermittent acclimation. The aim of this study was therefore to investigate these changes. To achieve our objectives, nine subjects received intermittent acclimation at low pressure in a barometric chamber (8?h daily for 5 days, day 1 at 4500 m, day 5 at 8500 m) before an expedition to the Himalayas. Hormonal and biochemical changes were studied using samples of venous blood taken at sea level before and after acclimation, after return from the expedition and 1 and 2 months after descent. Concentrations of thyroid hormones, adrenaline, noradrenaline (NA), hormones of hydromineral metabolism (aldosterone, renin, arginine vasopressin, atrial natriuretic peptide) as well as prolactin, cortisol, insulin and endothelin 1 were measured. Biochemical measurements made were plasma osmolality, and concentrations of glucose, total cholesterol, total proteins, pre-albumin, transferrin, complement 3C, apolipoproteins A₁ and B and serum iron. Acclimation induced no alteration in hormone (except for NA with increases of about 1.5, fold P<0.05) and biochemistry data. After the expedition, hormone responses were characterized by a higher total triidothyronine concentration (+18%, P<0.05) while other hormones did not vary. A linear relationship was found between thyroid-stimulating-hormone and body mass changes after the expedition (r=0.67, P<0.05). The observed increased concentrations of plasma proteins and total cholesterol (P<0.05) could be related to the restoration of lean body mass. At 1 and 2 months after return, no changes in hormones were observed but a significant decrease in transferrin concentration was noticed. The higher serum iron concentration reported after 1 month (P<0.05) could have been the result of a physiological haemolysis. It was concluded that both acclimation and the expedition in the Himalayas affected hormone status and body biochemistry status even though the observed changes were slight and rapidly reversed.