Verification of the two-dimensional disector, a method for the unbiased estimation of density and number of myelinated nerve fibers in peripheral nerves

Geuna, Stefano; Tos, Pierluigi; Battiston, Bruno; Guglielmone, R

doi:10.1016/s0940-9602(00)80117-x

Cited by 105 publications

(139 citation statements)

References 36 publications

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“…Stereology was carried out on a DM4000B microscope equipped with a DFC320 digital camera and an IM50 image manager system (Leica Microsystems, Wetzlar, Germany). Systematic random sampling and D-disector were adopted using a protocol previously described ( Geuna et al, 2000( Geuna et al, , 2004. Fiber density and total number of myelinated fibers were estimated together with fiber and axon diameter and myelin thickness.…”

Section: Sciatic Nerve Stereologymentioning

confidence: 99%

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

et al. 2012

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Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorbs membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorbs membrane, the [Ca 2þ ]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorbs membrane proved to be adequate and used as scaffold associated with undiffer-entiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorbs membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL).Stereological analysis was carried out on regenerated nerve fibers. In vitro investigation showed the formation of typical neuroglial cells after differentiation, which were positively stained for the typical specific neuroglial markers such as the GFAP, the GAP-43 and NeuN.NMR showed clear evidence that HMSCs expansion is glycolysis-dependent but their differentiation requires the switch of the metabolic profile to oxidative metabolism. In vivo studies showed enhanced recovery of motor and sensory function in animals treated with transplanted undifferentiated and differentiated HMSCs that was accompanied by an increase in myelin sheath. Taken together, HMSC from the umbilical cord Wharton jelly might be useful for improving the clinical outcome after peripheral nerve lesion.

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Section: Sciatic Nerve Stereologymentioning

confidence: 99%

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

et al. 2012

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“…Total cross‐sectional area (20× magnification), total fiber number (100× magnification), and mean fiber density were determined using a two‐dimensional procedure (optical fractionator; grid size: 150 × 150 μm²; counting frame size: 30 × 30 μm²) with the help of Stereo Investigator version 11.04 (MBF Bioscience, USA, RRID:SCR_002526). Only the fiber “tops” were counted to cope with the “edge effect” (Geuna, Tos, Battiston, & Guglielmone, 2000). In four randomly selected photomicrographs (100× magnification) of each section, axon and fiber diameter, myelin thickness, and g‐ratio (G‐ratio plug‐in, RRID:SCR_015580, http://gratio.efil.de/, ImageJ version 1.48, National Institutes of Health, USA, RRID:SCR_003070, 10 axons per picture, 80 examined axons per specimen, 320 considered axons per group and time point) were determined.…”

Section: Methodsmentioning

confidence: 99%

Reflex‐based grasping, skilled forelimb reaching, and electrodiagnostic evaluation for comprehensive analysis of functional recovery—The 7‐mm rat median nerve gap repair model revisited

2017

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IntroductionThe rat median nerve injury and repair model gets increasingly important for research on novel bioartificial nerve grafts. It allows follow‐up evaluation of the recovery of the forepaw functional ability with several sensitive techniques. The reflex‐based grasping test, the skilled forelimb reaching staircase test, as well as electrodiagnostic recordings have been described useful in this context. Currently, no standard values exist, however, for comparison or comprehensive correlation of results obtained in each of the three methods after nerve gap repair in adult rats.MethodsHere, we bilaterally reconstructed 7‐mm median nerve gaps with autologous nerve grafts (ANG) or autologous muscle‐in‐vein grafts (MVG), respectively. During 8 and 12 weeks of observation, functional recovery of each paw was separately monitored using the grasping test (weekly), the staircase test, and noninvasive electrophysiological recordings from the thenar muscles (both every 4 weeks). Evaluation was completed by histomorphometrical analyses at 8 and 12 weeks postsurgery.ResultsThe comprehensive evaluation detected a significant difference in the recovery of forepaw functional motor ability between the ANG and MVG groups. The correlation between the different functional tests evaluated precisely displayed the recovery of distinct levels of forepaw functional ability over time.ConclusionThus, this multimodal evaluation model represents a valuable preclinical model for peripheral nerve reconstruction approaches.

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“…These techniques are reliable, since they are designed to obtain unbiased quantitative results of the size, shape, and number of many biological objects. When these methods are properly used, they provide important contributions to experiments in biological research [9]. In this respect, our results may indicate real changes in the tissue and our comments on the nerve fibre morphology would be valid.…”

Section: Discussionmentioning

confidence: 55%

Original article Prenatal exposure of diclofenac sodium affects morphology but not axon number of the median nerve of rats

Ayrancı

Altunkaynak²,

Aktaş³

et al. 2013

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Verification of the two-dimensional disector, a method for the unbiased estimation of density and number of myelinated nerve fibers in peripheral nerves

Cited by 105 publications

References 36 publications

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

Reflex‐based grasping, skilled forelimb reaching, and electrodiagnostic evaluation for comprehensive analysis of functional recovery—The 7‐mm rat median nerve gap repair model revisited

Original article Prenatal exposure of diclofenac sodium affects morphology but not axon number of the median nerve of rats

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