Tissue-Clearing Technique and Cutaneous Nerve Biopsies: Quantification of the Intraepidermal Nerve-Fiber Density Using Active Clarity Technique-Pressure Related Efficient and Stable Transfer of Macromolecules Into Organs

BACKGROUND AND PURPOSE: Cutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies. METHODS: Five healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD). RESULTS: The mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm², respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm², respectively, and 3.06, 2.87, and 47.21 fibers/mm², respectively, in the patients with PHN. CONCLUSIONS: This study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

Establishing mouse model of type 2 diabetes peripheral neuropathy and measuring its intra-epidermal nerve fiber density / 临床麻醉学杂志

Objective To establish mouse model of type 2 diabetes peripheral neuropathy and measure its intra-epidermal nerve fiber density (IENFD).Methods Male C57BL6 mouse were ran-domly divided into four groups:group HS (n =6):high-fat diet+single streptozotocin intraperitoneal injection (120 mg/kg);group H (n =6):high fat diet+buffer injection;group S (n =6):standard chow diet+single streptozotocin intraperitoneal injection (120 mg/kg);group C (n = 6 ):standard chow diet+buffer injection.The 24th week was the end point of the experiment,and random glucose, homeostasis model assessment of insulin resistance (HOMA-IR),mechanical threshold,and IENFD were measured.Results Group HS had significantly higher random glucose and HOMA-IR than other groups (P <0.01),had significantly lower mechanical threshold than other groups (P <0.05), had significantly lower IENFD than groups S and C at 24th week (P <0.05 );group H had signifi-cantly higher random glucose and HOMA-IR than group C at 24th week (P <0.01),had no signifi-cant difference in mechanical threshold compared with group S and group C,and had no significant difference in IENFD compared with group HS.Conclusion A mouse model of type 2 diabetes periph-eral neuropathy was successfully established,and the IENFD was found to be decreased significantly.

Application of diffusion tensor tractography in grading gliomas / 实用放射学杂志

Objective To explore the value of diffusion tensor tractography (DTT)in grading gliomas.Methods 27 patients with brain glioma(gradeⅠ-Ⅱ in 1 1 cases and grade Ⅲ-Ⅳ in 1 6 cases)confirmed by pathology were collected.Conventional MR and DTT were carried out and the bilateral corticospinal tracts(CST)were reconstructed before operation.The fiber density index (FDi)and relative FDi (rFDi= ipsilateral FDi/contralateral FDi)of CST was measured.Results The FDi of ipsilateral CST was lower than that of the contralateral CST in all patients(P 0.05).The rFDi of LGG was higher than that of HGG (P <0.05).Conclusion In com-bination with conventional MR,DTT can improve the accuracy of grading gliomas.

Changes in corneal nerve fiber length and density before and after eye opening in mouse and its significance / 中华实验眼科杂志

Background Recently,there were many studies on corneal innervations during mammalian development.However,there were fewer studies on discussing corneal innervations before and after mouse eye openings.Objective The present study was to investigate the change in the regulation of corneal nerve fiber length and density before and after mouse eye openings to offer a basis for clinical research in human.Methods Thirty SPF C57BL/6 mice were divided into postnatal 1 day(P1 d),P7 d,P13 d(1 day before eye opening),P14 d(eye halfopened),P17 d(1 day after eye opening)and P23 d(7 day after eye opening)groups,with 5 mice and 10 eyes for each group.Entire corneal stretches were prepared and immunostaining with an anti-neuron-specific β-Ⅲ tubulin antibody was performed to label the corneal nerve fibers.Confocal microscopic pictures from the corneal dorsal-nasal region (DN),dorsal-temporal(DT),ventral-nasal region(VN)and ventral-temporal(VT)were taken using Delta Vision Core.From these pictures,the mouse corneal area,total length and density of nerve fibers in the 4 regions were calculated.The use of the animals complied with Statement of ARVO.Results Corneal areas of P1 d,P7d,P13 d,P14 d,P17 d and P23 d mice were(0.404±0.007),(1.362±0.154),(1.573±0.080),(1.603±0.046),(1.847±0.052),(2.445±0.798)mm2,respectively ; the total lengths of nerve fibers were(3.718±1.044),(19.065±3.350),(23.687±0.907),(27.309±2.477),(31.989±3.976),(41.214±1.573)mm,respectively ; the densities of nerve fibers were(9.592±1.138),(14.506±1.908),(15.088±1.241),(16.772±1.897),(16.821±2.102),(17.660±1.216)mm/mm2,respectively,all showing significant increases with age(F =22.906,P =0.000 ; F =0.424,P =0.000 ; F =2.375,P=0.000).A positive correlation of the increasing corneal areas and increasing lengths of nerve fibers was found(r=0.983,P＜0.01).Nerve fiber densities in the four corneal regions significantly increased with age(DN region:F =0.159,P =0.000 ; DT region:F =2.1 72,P =0.001 ; VN region:F =1.998,P =0.000 ; VT region:F=2.352,P=0.000).From P13 d to P14 d,the corneal nerve fiber densities in the DN region decreased by 6.0％ without significant difference(t =0.589,P =0.572); and the corneal nerve fiber densities in the DT region,VN region and VT region decreased by 4.6％,5.5％ and 0.1％,respectively,without significant difference from P14 d to P17 d(t=0.549,P=0.596;t=0.701,P=0.501 ;t=-0.100,P=0.919).Conclusions The development of nerve fibers in the whole cornea or the four corneal regions is influenced by eye opening in mouse to various extents.From P13 d to P14 d,the corneal nerve fiber densities in the DN region decreased by 6.0％ without significant difference.From P14 d to P17 d,the corneal nerve fiber densities in the DT region,VN region and VT region decrease by 4.6％,5.5％ and 0.1％,respectively,without significant difference.Afterwards,the growth of nerve fibers increased in pace and the growth rate is recovered.

MUSCULAR UNIT SIZE AND FIBER DENSITY DEDUCED FROM SIMULATION OF INVERSE ANALYSIS OF SURFACE ELECTROMYOGRAMS / 体力科学

The purpose of this study was to noninvasively extract information about the size and muscle fiber density of muscular units through the inverse analysis of surface electromyograms. Surface motor unit action potentials (MUAPs) were recorded with a multi-channel electrode array arranged along the circumference direction of the biceps brachii. The depth and intensity of equivalent current dipoles were estimated through the inverse analysis of surface MUAPs. The simulation of inverse analysis of surface potentials generated by the muscular unit models showed that the relationship between the depth and the intensity depends on the muscular unit size and muscle fiber density.<BR>In the simulation, we systematically varied the model parameters including distance from the skin, radius, and fiber density and used the inverse analysis to estimate the depth and intensity of current dipoles. And, our method to estimate the radius and fiber density of muscular units using estimated depth and intensity is demonstrated. Mean values (± SD) estimated from the surface MUAPs were 3.0±1.8mm for depth and 13.8±32.0nAm for intensity. The estimated distance ranged from slightly less than 1 mm to slightly more than 2 mm. The estimated radius ranged from 1.8 to 4.6 mm and fiber density from 0.7 to 5.4 fibers/mm<SUP>2</SUP>.

Diagnostic value of single fiber electromyography in diabetic peripheral neuropathy / 临床神经病学杂志

Objective To study the diagnostic value of single fiber electromyography (SFEMG) in diabetic peripheral neuropathy (DPN).Methods The values of jitter and fiber density (FD) in 36 patients with diabetes mellitus of 2 type were determined by Viking Ⅳ, while conventional nerve conduction studies (NCS) were performed,and the fasting blood glucose and glycosylated hemoglobin A 1C (HbA 1C ) were measured.Results The values of jitter were associated with FD, and both jitter and FD were positively related to HbA 1C .18 cases with abnormal NCS showed increased jitter(11 cases with block),and increased FD in 14 cases; the remaining 18 cases with normal NCS,7 cases with increased jitter(3 cases with block),the increased FD in 5 cases.Conclusion Denervation reinnervation reflected by jitter and FD was associated with the metabolic status. SFEMG could be served as sensitive means to early diagnosis of DPN, and might detect subclinical peripheral neuropathy of diabetes.