In vivo determination of normal and anterior cruciate ligament-deficient knee kinematics

The objective of the current study was to use fluoroscopy to accurately determine the three-dimensional (3D), in vivo, weight-bearing kinematics of 10 normal and five anterior cruciate ligament deficient (ACLD) knees. Patient-specific bone models were derived from computed tomography (CT) data. 3D computer bone models of each subject's femur, tibia, and fibula were recreated from the CT 3D bone density data. Using a model-based 3D-to-2D imaging technique registered CT images were precisely fit onto fluoroscopic images, the full six degrees of freedom motion of the bones was measured from the images. The computer-generated 3D models of each subject's femur and tibia were precisely registered to the 2D digital fluoroscopic images using an optimization algorithm that automatically adjusts the pose of the model at various flexion/extension angles. Each subject performed a weight-bearing deep knee bend while under dynamic fluoroscopic surveillance. All 10 normal knees experienced posterior femoral translation of the lateral condyle and minimal change in position of the medial condyle with progressive knee flexion. The average amount of posterior femoral translation of the lateral condyle was 21.07 mm, whereas the average medial condyle translation was 1.94 mm, in the posterior direction. In contrast, all five ACLD knees experienced considerable change in the position of the medial condyle. The average amount of posterior femoral translation of the lateral condyle was 17.00 mm, while the medial condyle translation was 4.65 mm, in the posterior direction. In addition, the helical axis of motion was determined between maximum flexion and extension. A considerable difference was found between the center of rotation locations of the normal and ACLD subjects, with ACLD subjects exhibiting substantially higher variance in kinematic patterns.     

In vivo hybridisation of cultured melanoma cells and isogenic normal mouse cells.

Somatic cell hybrids were derived by fusing tumourigenic and melanogenic melanoma (PAZG) cells with normal diploid male mouse cells in vivo. Their chromosomal composition was equivalent to the sum of both parental genomes and included a Y chromosome lacking in the melanoma parent. Our study showed that in PAZG X C57BL hybrids (MP), tumourigenicity was suppressed but pigmentation was expressed.     

In vivo and in vitro lysis of mouse cancer cells by antimetastatic effectors in normal plasma

Summary Results of parallel in vivo (IV. challenge) and in vitro (trypan blue staining) tests suggest that normal molecular factors in mouse blood can rapidly lyse malignant cells that enter the circulation. An inverse relationship was seen between the ability of malignant cells to form metastases after IV. injection and their susceptibility to lysis in vivo and in vitro. A mammary carcinoma lost its susceptibility to lysis and gained an ability to form metastases with conversion to the ascites form. The factor(s) active against tumor cells in vitro was were most evident in whole plasma, less so in serum form. The factors active against tumor cells in vitro globulin fraction of normal serum. Whole serum from tumor hosts had lower activity than whole normal serum, but had gained some activity in the immunoglobulin fraction. Treatment of normal animals with immunogens reduced the antitumor activity of whole serum. Whole-body irradiation, and, to a lesser degree, cyclophosphamide treatment, increased the activity.     

Regional distribution of protein kinases in normal and odor-deprived mouse olfactory bulbs

Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKAalpha), protein kinase C (PKCalpha), and CaM kinases II (CaMKIIalpha, beta) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKAalpha immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKCalpha staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKAalpha, PKCalpha and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels.     

In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2′-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley-Liss, Inc.     

In vivo recordings of spontaneous and odor-modulated dynamics in the Limax olfactory lobe

The major central site of olfactory information processing in the terrestrial slug Limax maximus is the procerebral lobe of the cerebral ganglion, which exhibits oscillatory dynamics of its local field potential and propagates activity waves from its apex to its base, as determined by multisite optical and electrical measurements in vitro. The learning-dependent uptake of Lucifer yellow into procerebral neurons suggests that the procerebral lobe may form learned representations of odors. To determine the role of the procerebral lobe in odor processing and odor learning, we developed procedures to implant fine wire electrodes in the lobe, which allowed recordings of local field potential in freely behaving slugs. The procerebral lobe displays oscillatory dynamics of its local field potential in vivo; however the amplitude and frequency of the local field potential are much more variable in vivo than in vitro. Odor presentation leads to increased frequency and amplitude of the local field potential signal. Several lines of evidence indicate that the variations in the local field potential signal recorded in vivo are not due to movement artifacts or activity in adjacent muscles. Multiple amine, gaseous, and peptide neuromodulators known to be present in the procerebral lobe provide pathways by which activity or coupling of bursting neurons in the procerebral lobe could be altered, resulting in the observed amplitude and frequency modulation of the local field potential.     

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.     

In vitro studies on the determination of the coagulating activity of normal, inflammatory and bloody cerebrospinal fluids

Selective analyses of the coagulation factors II, V, VIII, and X in cerebrospinal fluid resulted in activity values ranging from 0.01-0.05 as a portion of the relative ratio 1 pertaining to the plasma activity. The increase of activity did not correlate with the protein increase in cerebrospinal fluid at F-VIII, and only to a slight extent at F-II, F-V,, and F-X. As compared with plasma a stimulating effect on coagulation can be recorded thromboelastographically in plasma mixtures.     

活体电转化技术在新生小鼠视网膜中的应用

活体电转化技术是在高电压的脉冲作用下,瞬态增加细胞膜的渗透性从而将外源基因高效导入细胞的方法。与病毒载体等其他方法相比,活体电转化技术具有安全、高效、快速、稳定及应用范围广等优点,近年来在很多组织和器官中得到广泛使用,包括在眼科研究领域。文章介绍了活体电转化技术在新生小鼠视网膜中的应用,通过新生小鼠视网膜下注射的方法,经几次高电压的脉冲,将高浓度的绿色荧光蛋白表达质粒导入新生小鼠视网膜细胞内。通过冰冻切片观察绿色荧光蛋白在视网膜中的表达。结果表明绿色荧光蛋白在视网膜外核层高表达,证实了活体电转化技术可以将外源基因高效、快捷的导入视网膜,从而为研究视网膜发育及功能提供一种有效的手段。     

In vivo estimation of tracheal distensibility and hysteresis in normal adults

We used the acoustic reflection technique to measure the cross-sectional area of tracheal and bronchial airway segments of eight healthy adults. We measured airway area during a slow continuous expiration from total lung capacity (TLC) to residual volume (RV) and during inspiration back to TLC. Lung volume and esophageal pressure were monitored continuously during this quasi-static, double vital capacity maneuver. We found that 1) the area of tracheal and bronchial segments increases with increasing lung volume and transpulmonary pressure, 2) the trachea and bronchi exhibit a variable degree of hysteresis, which may be greater or less than that of the lung parenchyma, 3) extrathoracic and intrathoracic tracheal segments behaved as if they were subjected to similar transmural pressure and had similar elastic properties, and 4) specific compliance (means +/- SE) for the intrathoracic and bronchial segments, calculated with the assumption that transmural pressure is equal to the transpulmonary pressure, was significantly (P less than 0.05) smaller for the intrathoracic segment than for the bronchial segment: (2.1 +/- 2.0) X 10(-3) cmH2O-1 vs. (9.1 +/- 2.1) X 10(-3) cmH2O-1. Direct measurements of airway area using acoustic reflections are in good agreement with previous estimates of airway distensibility in vivo, obtained by radiography or endoscopy.     

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.     

In vivo transfection of the mouse vas deferens

To explore the possibility that specific characteristics of the epithelium of the male tract can be modified, transfections of the mouse vas deferens have been performed using in vivo injections of cationic DNA/liposome complexes. Gene transfer was done employing the reporter genes pEGFP-C1 encoding Green Fluorescent Protein (GFP) and pCMV-nls-beta encoding the nuclear beta-Galactosidase (beta-Gal). Foreign gene expression reached a maximum of 6.8% in the epithelial cells of the vas after treatment with the nuclear beta-Gal gene construction and of 13.3% after employing the GFP gene construction. Expression of the GFP gene appeared from one week up to three months following injection, and it appeared as patches of modified cells along the epithelium. Results from immunocytochemistry and Western Blotting support the conclusion that transfection of epithelial cells was achieved. We have also transfected the vas using gene constructions that express secretory proteins--specifically, the reporter system pSEAP-control that expresses a secretory form of human placental alkaline phosphatase, and the pGFP-Ctk-37 that expresses a secretion form of GFP. In both cases, the fluids expressed from the transfected vas showed a significant increase of alkaline phosphatase activity after pSEAP transfection and the presence of GFP protein when pGFP-Ctk-37 gene construction was employed. Our results indicate that the vas can be transfected in vivo using liposomes as vectors of foreign genes and that the vas fluid contents can be modified.     

In vivo interaction between mouse liver lysosomes and chloroquine

Particles sedimenting at 27,000 g X 10 min (MLCQ) were separated from liver homogenates of mice injected with chloroquine (CQ). The MLCQ contained most of the drug recovered in the organ as well as 50% of the liver aryl sulphatase activity. The release of CQ from MLCQ was studied in some physicochemical conditions, and in the presence of various agents known to modify membrane composition and stability. At pH 7.4, the equilibrium between free and bound CQ depended on the dilution of the MLCQ, and the time to reach equilibrium was strongly influenced by the temperature of incubation. Several agents causing membrane disruption and lysosomal enzyme leakage, such as osmotic shock, sonication and digitonin, had little effect on the CQ release. Acid and alkaline buffers, 0.55 M KCl and 0.1% Triton X-100 caused, instead, the immediate release of most of the bound CQ. Concentrations of digitonin causing the release of aryl sulphatase activity had little effect on bound CQ, suggesting that the drug is retained in lysosomes by forces and/or structures different in nature from those retaining most of the lysosomal enzyme activity. We think that the CQ trapped in lysosomes is bound to high affinity sites in membranous structures which are particularly altered by agents known to extract peripheral proteins from biological membranes or to change the conformation of molecular structures.     

In vivo RNA-RNA recombination of coronavirus in mouse brain.

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.     

In vivo monitoring of peripheral circadian clocks in the mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.     

In vivo imaging of autophagy in a mouse stroke model

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.