Cleaning lateral morphological features of the root canal: the role of streaming and cavitation.

AIM: To investigate the effects of ultrasonic activation file type, lateral canal location and irrigant on the removal of a biofilm-mimicking hydrogel from a fabricated lateral canal. Additionally, the amount of cavitation and streaming was quantified for these parameters. METHODOLOGY: An intracanal sonochemical dosimetry method was used to quantify the cavitation generated by an IrriSafe 25 mm length, size 25 file inside a root canal model filled with filtered degassed/saturated water or three different concentrations of NaOCl. Removal of a hydrogel, demonstrated previously to be an appropriate biofilm mimic, was recorded to measure the lateral canal cleaning rate from two different instruments (IrriSafe 25 mm length, size 25 and K 21 mm length, size 15) activated with a P5 Suprasson (Satelec) at power P8.5 in degassed/saturated water or NaOCl. Removal rates were compared for significant differences using nonparametric Kruskal-Wallis and/or Mann-Whitney U-tests. Streaming was measured using high-speed particle imaging velocimetry at 250 kfps, analysing both the oscillatory and steady flow inside the lateral canals. RESULTS: There was no significant difference in amount of cavitation between tap water and oversaturated water (P = 0.538), although more cavitation was observed than in degassed water. The highest cavitation signal was generated with NaOCl solutions (1.0%, 4.5%, 9.0%) (P < 0.007) and increased with concentration (P < 0.014). The IrriSafe file outperformed significantly the K-file in removing hydrogel (P < 0.05). Up to 64% of the total hydrogel volume was removed after 20 s. The IrriSafe file typically outperformed the K-file in generating streaming. The oscillatory velocities were higher inside the lateral canal 3 mm compared to 6 mm from WL and were higher for NaOCl than for saturated water, which in turn was higher than for degassed water. CONCLUSIONS: Measurements of cavitation and acoustic streaming have provided insight into their contribution to cleaning. Significant differences in cleaning, cavitation and streaming were found depending on the file type and size, lateral canal location and irrigant used. In general, the IrriSafe file outperformed the K-file, and NaOCl performed better than the other irrigants tested. The cavitation and streaming measurements revealed that both contributed to hydrogel removal and both play a significant role in root canal cleaning.

The effect of deprivation on the incidence of mandibular fractures in a British city.

AIM: To examine the relationship between social and material deprivation and mandibular fractures. METHOD: Three hundred and forty three consecutive patients who underwent mandibular fracture fixation were selected for the study. After exclusions, 290 were divided into age groups and ranked according to their Index of Multiple Deprivation (IMD) score. Rankings were determined using postcodes, and divided into quintiles for statistical analysis. RESULTS: Ages ranged from 7 to 82 with 146 (50%) patients aged between 20 and 29. Males accounted for 85% of cases. The most common site of fracture was the angle (n = 195) and assault was shown to be the most common mechanism of injury (63.3%). A strong relationship was demonstrated between fractures of the mandible and worsening deprivation, with the most striking relationship seen with fractures sustained as a consequence of assault. Females were less likely than males to sustain a fracture of the mandible as a consequence of assault; however, when assault was the mechanism of injury they were also likely to be from a deprived background. CONCLUSION: This study has demonstrated that a strong relationship exists between deprivation and the incidence of mandibular fractures in our catchment area. Fractures that resulted from interpersonal violence were shown to have a particularly strong correlation with deprivation.

Shaping ability of single-file reciprocating and heat-treated multifile rotary systems: a micro-CT study.

AIM: To investigate changes in three-dimensional geometry, in various cross-sectional morphological parameters and in the centring ability of root canals prepared with different preparation systems using microcomputed tomographic imaging technology. METHODOLOGY: Sixty-four mesial canals of mandibular molars were matched based on similar morphological dimensions using micro-CT evaluation and assigned to four experimental groups (n = 16), according to the canal preparation technique: Reciproc, WaveOne, Twisted File and HyFlex CM systems. Changes in several 2D (area, perimeter, form factor, roundness, minor and major diameter) and 3D [volume, surface area, structure model index (SMI)] morphological parameters, as well as canal transportation, were compared with preoperative values using Kruskal-Wallis and anovapost hoc Tukey's tests with the significance level set at 5%. RESULTS: Preparation significantly increased all tested parameters in the experimental groups. No significant differences were observed between groups regarding changes in volume, surface area, SMI, form factor and roundness of the root canal after preparation (P > 0.05). In the apical third, the Reciproc group had significantly greater changes in canal area, perimeter, major and minor diameters than the other groups (P < 0.05). Overall, the Twisted File and HyFlex CM systems were associated with significantly less transportation than the reciprocating instruments, Reciproc and WaveOne (P < 0.05). CONCLUSIONS: Shaping procedures led to the enlargement of the root canal space with no evidence of significant preparation errors. Changes in 3D parameters were not different between groups whilst, in the apical third, Reciproc was associated with significantly greater changes in several 2D parameters compared to the other groups. Twisted File and HyFlex CM systems were able to maintain the original canal anatomy with less canal transportation than Reciproc and WaveOne; however, these differences are unlikely to be of clinical significance.

Medical supply on contingency military operations: experience from Operation GRITROCK.

Medical supply during military operations has the ability to affect the efficacy of the operation being undertaken, either negatively or positively. An appropriately-managed maritime platform with a robust medical supply chain during transit and on arrival in theatre is the main aim. A secure supply chain will reduce any implications that logistics may have with regard to capability, and negate the effects of deficiencies of short shelf life items occurring over time and during use in high tempo operations.

A novel methodology providing insights into removal of biofilm-mimicking hydrogel from lateral morphological features of the root canal during irrigation procedures.

AIM: To introduce and characterize a reproducible hydrogel as a suitable biofilm mimic in endodontic research. To monitor and visualize the removal of hydrogel from a simulated lateral canal and isthmus for the following: I) Ultrasonic-Activated Irrigation (UAI) with water, ii) UAI with NaOCl and iii) NaOCl without UAI. METHODOLOGY: A rheometer was used to characterize the viscoelastic properties and cohesive strength of the hydrogel for suitability as a biofilm mimic. The removal rate of the hydrogel from a simulated lateral canal or isthmus was measured by high-speed imaging operating at frame rates from 50 to 30,000 fps. RESULTS: The hydrogel demonstrated viscoelastic behaviour with mechanical properties comparable to real biofilms. UAI enhanced the cleaning effect of NaOCl in isthmi (P < 0.001) and both NaOCl and water in lateral canals (P < 0.001). A greater depth of cleaning was achieved from an isthmus (P = 0.009) than from a lateral canal with UAI and also at a faster rate for the first 20 s. NaOCl without UAI resulted in a greater depth of hydrogel removal from a lateral canal than an isthmus (P < 0.001). The effect of UAI was reduced when stable bubbles were formed and trapped in the lateral canal. Different removal characteristics were observed in the isthmus and the lateral canal, with initial highly unstable behaviour followed by slower viscous removal inside the isthmus. CONCLUSIONS: The biofilm-mimicking hydrogel is reproducible, homogenous and can be easily applied and modified. Visualization of its removal from lateral canal anatomy provides insights into the cleaning mechanisms of UAI for a biofilm-like material. Initial results showed that UAI improves hydrogel removal from the accessory canal anatomy, but the creation of stable bubbles on the hydrogel-liquid interface may reduce the cleaning rate.

Application of wavelet denoising to improve compression efficiency while preserving integrity of digital micrographs.

Modern microscopy methods require efficient image compression techniques owing to collection of up to thousands of images per experiment. Current irreversible techniques such as JPEG and JPEG2000 are not optimized to preserve the integrity of the scientific data as required by 21 CFR part 11. Therefore, to construct an irreversible, yet integrity-preserving compression mechanism, we establish a model of noise as a function of signal in our imaging system. The noise is then removed with a wavelet shrinkage algorithm whose parameters are adapted to local image structure. We ascertain the integrity of the denoised images by measuring changes in spatial and intensity distributions of registered light in the biological images and estimating changes of the effective microscope MTF. We demonstrate that the proposed denoising procedure leads to a decrease in image file size when a reversible JPEG2000 coding is used and provides better fidelity than irreversible JPEG and JPEG2000 at the same compression ratio. We also demonstrate that denoising reduces image artefacts when used as a pre-filtering step prior to irreversible image coding.

Validation of large-scale, monochromatic UV disinfection systems for drinking water using dyed microspheres.

Dyed microspheres have been developed as a new method for validation of ultraviolet (UV) reactor systems. When properly applied, dyed microspheres allow measurement of the UV dose distribution delivered by a photochemical reactor for a given operating condition. Prior to this research, dyed microspheres had only been applied to a bench-scale UV reactor. The goal of this research was to extend the application of dyed microspheres to large-scale reactors. Dyed microsphere tests were conducted on two prototype large-scale UV reactors at the UV Validation and Research Center of New York (UV Center) in Johnstown, NY. All microsphere tests were conducted under conditions that had been used previously in biodosimetry experiments involving two challenge bacteriophage: MS2 and Qbeta. Numerical simulations based on computational fluid dynamics and irradiance field modeling were also performed for the same set of operating conditions used in the microspheres assays. Microsphere tests on the first reactor illustrated difficulties in sample collection and discrimination of microspheres against ambient particles. Changes in sample collection and work-up were implemented in tests conducted on the second reactor that allowed for improvements in microsphere capture and discrimination against the background. Under these conditions, estimates of the UV dose distribution from the microspheres assay were consistent with numerical simulations and the results of biodosimetry, using both challenge organisms. The combined application of dyed microspheres, biodosimetry, and numerical simulation offers the potential to provide a more in-depth description of reactor performance than any of these methods individually, or in combination. This approach also has the potential to substantially reduce uncertainties in reactor validation, thereby leading to better understanding of reactor performance, improvements in reactor design, and decreases in reactor capital and operating costs.

Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes.

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells.

HBEGF promotes gliomagenesis in the context of Ink4a/Arf and Pten loss.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is a ligand for the EGF receptor (EGFR), one of the most commonly amplified receptor tyrosine kinases (RTKs) in glioblastoma (GBM). While HBEGF has been found to be expressed in a subset of malignant gliomas, its sufficiency for glioma initiation has not been evaluated. In this study, we demonstrate that HBEGF can initiate GBM in mice in the context of Ink4a/Arf and Pten loss, and that these tumors are similar to the classical GBM subtype observed in patients. Isogenic astrocytes from these mice showed activation not only of Egfr but also the RTK Axl in response to HBEGF stimulation. Deletion of either Egfr or Axl decreased the tumorigenic properties of HBEGF-transformed cells; however, only EGFR was able to rescue the phenotype in cells lacking both RTKs indicating that Egfr is required for activation of Axl in this context. Silencing of HBEGF in vivo resulted in tumor regression and significantly increased survival, suggesting that HBEGF may be a clinically relevant target.

Activated MEK cooperates with Cdkn2a and Pten loss to promote the development and maintenance of melanoma.

The development of targeted inhibitors, vemurafenib and dabrafenib, has led to improved clinical outcome for melanoma patients with BRAFV600E mutations. Although the initial response to these inhibitors can be dramatic, sometimes causing complete tumor regression, the majority of melanomas eventually become resistant. Mitogen-activated protein kinase kinase (MEK) mutations are found in primary melanomas and frequently reported in BRAF melanomas that develop resistance to targeted therapy; however, melanoma is a molecularly heterogeneous cancer, and which mutations are drivers and which are passengers remains to be determined. In this study, we demonstrate that in BRAFV600E melanoma cell lines, activating MEK mutations drive resistance and contribute to suboptimal growth of melanoma cells following the withdrawal of BRAF inhibition. In this manner, the cells are drug-addicted, suggesting that melanoma cells evolve a 'just right' level of mitogen-activated protein kinase signaling and the additive effects of MEK and BRAF mutations are counterproductive. We also used a novel mouse model of melanoma to demonstrate that several of these MEK mutants promote the development, growth and maintenance of melanoma in vivo in the context of Cdkn2a and Pten loss. By utilizing a genetic approach to control mutant MEK expression in vivo, we were able to induce tumor regression and significantly increase survival; however, after a long latency, all tumors subsequently became resistant. These data suggest that resistance to BRAF or MEK inhibitors is probably inevitable, and novel therapeutic approaches are needed to target dormant tumors.

Deficient axonal transport of substance P in streptozocin-induced diabetic rats. Effects of sorbinil and insulin.

This study measured the accumulation of substance P-like immunoreactivity (SPLI) proximal and distal to 12-h constricting ligatures applied to rat sciatic nerves. There were three separate experiments, and the baseline for each consisted of control and age-matched rats with 3 wk of untreated streptozocin-induced diabetes. We compared the effects of twice-daily insulin treatment, daily sorbinil (25 mg.kg-1.day-1 p.o.), and a combination of both treatments. In untreated diabetic rats the anterograde accumulation of SPLI was reduced by 30-40%. This deficit was unaffected by sorbinil alone but was attenuated by insulin and prevented completely by insulin and sorbinil combined. There were also indications that diabetes caused reductions in retrograde accumulation of SPLI and its content in unconstricted nerve and the L4 dorsal root ganglion. The fraction of SPLI undergoing net anterograde or retrograde movement and the velocities of accumulation were unaffected by diabetes or the treatment regimens. These findings indicate a reduction in the amount of substance P moved by axonal transport in diabetic rats that is related partly to aldose reductase activity and partly to some other insulin-correctable consequence of experimental diabetes.

Three-dimensional structural analysis of tetanus toxin by electron crystallography.

Two-dimensional crystalline arrays of native tetanus toxin have been formed at the interface between a solution of the toxin and a phospholipid monolayer containing a ganglioside. Electron crystallographic analysis has been used to study these periodic arrays. The arrays obey the symmetry of plane group p12(1), with a = 126 A and b = 84 A, and a thickness of 90 A (1 A = 0.1 nm). The three-dimensional structure of tetanus toxin in negative stain is reconstructed to a nominal resolution of 14 A from multiple tilt images. The molecule presents an asymmetric three-lobed structure and could interact with the monolayer in two possible orientations.

Ca2+ redistribution from bound to free form is required for tumor necrosis factor actions in 30A5 preadipocytes.

Tumor necrosis factor/cachectin (TNF) inhibits differentiation of 30A5 preadipocytes into adipocytes. In this process, TNF inhibits the expression of the gene for acetyl-coenzyme-A carboxylase, the rate-limiting enzyme for biogenesis of long chain fatty acids. One of the early reactions caused by TNF is the Ca2+ redistribution of Ca2+ from the bound form to the free form. This Ca2+ redistribution results in a transient Ca2+ efflux. High concentrations of Mg2+ inhibit Ca2+ redistribution and efflux. This inhibition reverses the repression of acetyl-coenzyme-A carboxylase and reverses the TNF inhibition of the differentiation of 30A5 preadipocytes into adipocytes. This indicates that Ca2+ redistribution between the bound and the free form is an obligatory event in the sequence of actions caused by TNF in 30A5 cells.

Defects in intracellular oxidative metabolism of neutrophils undergoing apoptosis.

Apoptosis permits neutrophil recognition by macrophages, thereby not only limiting potential cytotoxicity but also promoting resolution of inflammation. A direct relationship between apoptosis and intracellular hydrogen peroxide (H2O2) production was observed in phorbol 12-myristate 13-acetate (PMA) -stimulated neutrophils aged in culture. A significant decrease in intracellular H2O2 production was observed in aging neutrophils at 12, 24, and 48 h. However, intracellular superoxide anion production in response to PMA stimulation was preserved up to 24 h, implying retention of intracellular signaling pathways leading to NADPH oxidase stimulation. A significant decrease in the cytoplasmic content and activity of Cu,Zn superoxide dismutase was responsible for the observed decline in intracellular H2O2 production in apoptotic neutrophils. Intracellular glutathione content also decreased concomitantly with H2O2 production. These observations indicate that onset of apoptosis in neutrophils is in part mediated by oxidative stress resulting from the down-regulation of key antioxidant defense systems of the cell, namely superoxide dismutase and glutathione.

Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.

One of the objectives of studying endothelial cells in vitro is to evaluate neutrophil-endothelial cell interactions including potential consequences of oxidant-mediated damage to the endothelial cell. Current understanding of endothelial cell oxidative function is derived primarily from the measurement of extracellular products. We utilized 2 dyes, 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), which measure hydrogen peroxide (H2O2) and superoxide anion (O2-) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH-DA and stimulated with H2O2 exhibited an increase in the fluorescent product 2',7'-dichlorofluorescein (DCF) (measure of intracellular H2O2) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H2O2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O2-) which lasted for approximately 60 min. Superoxide dismutase increased DCF fluorescence in endothelial cells stimulated with H2O2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduced DCF and EB fluorescence by 48% and 37% respectively in endothelial cells stimulated with H2O2. Catalase completely inhibited an increase in DCF or EB fluorescence in endothelial cells stimulated with H2O2. There was a direct correlation between mean DCF and EB fluorescence intensity and the concentration of H2O2 or the number of phorbol 12-myristate 13-acetate-activated neutrophils added to endothelial cells. We conclude from these studies that DCFH-DA and HE can be used to measure intracellular H2O2 and O2- in endothelial cells and that the xanthine oxidase pathway for intracellular O2- production accounts for approximately 40% of the total intracellular O2- generated in endothelial cells after stimulation with H2O2. The combination of image cytometry and flow cytometry will be important for future evaluations of endothelial cell function.

Measurement of intracellular fluorescence of human monocytes relative to oxidative metabolism.

Human monocytes (MN) produce O2- and H2O2 when stimulated by agonists. Dichlorofluorescin diacetate (DCFH-DA) has been used as a substrate for measuring intracellular oxidant production in neutrophils. DCFH-DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. DCFH-DA can not be appreciably oxidized to a fluorescent state without prior hydrolysis. We have examined the utility of DCFH-DA for the assessment of monocyte oxidative responses. The levels of intracellular fluorescence measured by flow cytometry were considerably less than expected from reported levels of O2--production or chemiluminescence assays. Compared with neutrophils, monocytes produced minimal increases in DCF fluorescence after stimulation with phorbol myristate acetate as measured by flow cytometry, but both cell types showed increases in fluorescence when bulk cell suspensions were measured by spectrofluorometry. To determine the intracellular location of the DCFH, bulk fluorescence measurements were made on both whole and sonicated cell preparations. When intact mononuclear cells were preloaded with DCFH-DA, then sonicated and oxidized with added excess H2O2, the increase in fluorescence was only 30% of the fluorescence of mononuclear cell sonicates to which DCFH-DA was added and oxidized in a similar manner. These results suggest that a portion of the DCFH-DA incorporated by intact cells, is not susceptible to oxidation by the added H2O2. Addition of NaOH to induce hydrolysis of any residual DCFH-DA in the sonicates of DCFH-DA-loaded intact mononuclear cells resulted in a further increase in fluorescence upon addition H2O2, suggesting that a significant portion of the DCFH-DA was not hydrolyzed despite ample uptake of this dye by these cells. In contrast, no further increase in fluorescence was observed in sonicates of DCFH-DA-loaded intact neutrophils, suggesting complete hydrolysis of all incorporated DCFH-DA to DCFH. When monocytes were allowed to phagocytose DCFH-DA-loaded Staphylococcus aureus, intracellular fluorescence was measurable by flow cytometry, indicating intracellular oxidation of the fluorochromes. We therefore propose that in monocytes the mechanism of intracellular processing of these fluorochromes differs from that in neutrophils owing to differences in intracellular localization of fluorochromes, site of oxidant production, and/or accessibility of the DCFH-DA to esterolysis.

Impairment of human neutrophil oxidative burst by polychlorinated biphenyls: inhibition of superoxide dismutase activity.

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O2- to H2O2. Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular O2- production along with a significant decrease in H2O2 production compared with unexposed agonist-stimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L-methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-alpha. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzyme-mediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis.

Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Niguldipine discriminates between alpha 1-adrenoceptor-mediated second messenger responses in rat cerebral cortex slices.

The effect of both isomers of niguldipine, a highly selective alpha 1-adrenoceptor antagonist and dihydropyridine calcium channel blocker, on noradrenaline-stimulated inositol phosphate (IP) accumulation and adenosine 3':5'-cyclic monophosphate (cyclic AMP) potentiation was examined. Both isomers inhibited noradrenaline-stimulated IP accumulation. (+)-Niguldipine was 100 fold more potent than (-)-niguldipine. Potentiation of beta-adrenoceptor-stimulated cyclic AMP by noradrenaline was only partially inhibited by both isomers. The dihydropyridine, israpidine, did not inhibit either second messenger response. This study provides further evidence that the alpha 1-adrenoceptors mediating IP accumulation and cyclic AMP potentiation are different.

Prevention of defects of axonal transport in experimental diabetes by aldose reductase inhibitors.

Experiments on streptozotocin-diabetic rats have indicated that axonal transport of choline acetyltransferase is reduced in sciatic nerve. Treatment with an aldose reductase inhibitor both prevented and reversed this defect which was related to marked accumulations of sorbitol and fructose. Concurrent with these accumulations the content of myo-inositol in diabetic peripheral nerve is depleted. Further experiments taking account of nerve water content showed that the depletion of myo-inositol was 'real' not apparent. When the level of myo-inositol was maintained, either by feeding myo-inositol or by the inhibition of aldose reductase, the development of defective axonal transport of choline acetyltransferase and choline-containing lipids was prevented.