Geuna, Stefano

Gruessinger, Hannes

Jaminet, Patrick

Manoli, Theodora

Schaller, Hans Eberhard

Schiefer, Jennifer Lynn

Sinis, Nektarios

Werdin, Frank

English

Institutional Research Information System University of Turin

Correlation Analysis of Histomorphometry andMotor Neurography in the Median Nerve RatModelTheodora Manoli, MD,a,∗ Frank Werdin, MD,a,∗ Hannes Gruessinger, MD,aNektarios Sinis, MD,a Jennifer Lynn Schiefer, MD,a Patrick Jaminet, MD,aStefano Geuna, MD,b and Hans-Eberhard Schaller, MDaaClinic of Hand, Plastic, and Reconstructive Surgery with Burn Unit, BG-Trauma Centre, Universityof Tuebingen, Schnarrenbergstr. 95, 72076 Tuebingen, Germany; and bDepartment of Clinical andBiological Sciences, University of Turin, San Luigi Hospital, Orbassano (TO), Azienda OspedalieraSan Luigi - Regione Gonzole 10, 10043 Turin, ItalyCorrespondence: theodora.manoli@gmail.com∗Both authors have contributed equally to this research article.Keywords: neurography, electrophysiology, morphometry, nerve regeneration, median nervePublished April 9, 2014Objective: Standard methods to evaluate the functional regeneration after injury of therat median nerve are insufficient to identify any further differences of axonal nerveregeneration after restitution of motor recovery is completed. An important comple-mentary method for assessing such differences is a histomorphometric analysis of thedistal to lesion nerve fibers. Recently, an electrophysiological method has been proposedas a sensitive method to examine the quality of axonal nerve regeneration. Methods:A linear regression analysis has been performed to correlate histomorphometric andneurographic data originating from 31 rats subjected to neurotmesis and immediatereconstruction of their right median nerve. Results: A significant linear correlation be-tween the velocity of neuromuscular conduction and the total number of nerve fibers(P = .037) as well as between the amplitude of compound muscle action potential andthe total number of nerve fibers (P= .026) has been identified. Interestingly, a significantcorrelation between the velocity of neuromuscular conduction and the square root of thecross-sectional area of the nerve could be found (P = .008). This corresponds to a linearcorrelation between the velocity of neuromuscular conduction and the radius of thenerve. Conclusion: These results contribute in a better interpretation of morphologicalpredictors of nerve regeneration and verify the previously described electrophysiologicalassessment in the median nerve rat model as a valid method.The median nerve model of the rat became a popular tool to examine peripheral nerveregeneration under several conditions in the last years. The functional recovery can besimply and reliably assessed by the so-called grasping test as well as by weighing the flexor136MANOLI ET ALdigitorum sublimis muscle.1 In most cases, the grasp force recovers almost completely inabout 3months after nerve lesion. A further and precious supplementary tool to quantify theaxonal regeneration is the histomorphometric analysis of the distal to lesion nerve segment.2With this method, parameters such as nerve cross-sectional area, total fiber number, fiberdensity, diameter of fibers and axons, and myelin thickness can be calculated.Recently, an electrophysiological method to perform a motor neurography in the me-dian nerve rat model has been established by our group.3 With this method, parameters likethe threshold to evoke a compound muscle action potential (CMAP), latency, CMAP andvelocity of neuromuscular transduction can be assessed by a standardized procedure. Thedevelopment of motor neurography for the median nerve rat model gave us the opportunityto get more information about the quality and extent of the axonal regeneration that hastaken place over several time points.The purpose of the actual study was to correlate electrophysiological parametersand histomorphological findings from the median nerve model of the rat, after functionalrecovery was completed. Furthermore, our results could validate both methods as moresensible tools to evaluate axonal regeneration, compared to the standard functional tests.MATERIALS AND METHODSData acquisitionThe electrophysiological and histomorphological data used for our correlation analysisoriginated from 2 already published works of our department.4,5 In both works, the me-dian nerve model of Wistar adult female rats, weighing 220 to 250 g each, was used. Intotal, 31 rats originating from these works were subjected to both electrophysiological andhistomorphological analysis 12 weeks after surgery. Experiments have been carried out inaccordance to EC Directive 86/609/EEC for animal experiments.The distribution of animals according to their treatment after neurotmesis of the rightmedian nerve is depicted in Table 1. The electrophysiological parameters assessed by ourstandard protocol3 and used for the actual correlation analysis have been as follows:– The threshold [V] of stimulus to provoke a CMAP– The transduction velocity or v [m/s], which was calculated by the distance betweenthe stimulus and the electrode placed in the flexor sublimis muscle divided by thelatency between the stimulus and the beginning of the CMAP, and– the amplitude of the CMAP [μV]Table 1. Distribution of animals according to their surgical treatment as described instudies A4 and B5Study Treatment Number of animalsA Direct suture 3A Direct suture plus vein-graft wrapping 4A Direct suture plus vein-graft wrapping filled with Perineurin vehicle 2A Direct suture plus vein-graft wrapping filled with Perineurin 6B Direct suture 5B FloSeal application to the nerve stumps and direct suture 6B Electrocoagulation of the nerve stumps and direct suture 5137ePlasty VOLUME 14Table 2. F-statistic of the binary regression analyses between the histomorpho-metric and neurographic dataNeurographic parametersThreshold Transduction velocity AmplitudeHistomorphometric parametersCross sectional area 0.562 0.009 0.083Total fiber number (N) 0.201 0.037 0.026Fiber density 0.406 0.133 0.869Fiber diameter (D) 0.543 0.749 0.556Axon diameter (d) 0.603 0.680 0.767Myelin thickness (M) 0.860 0.859 0.514G ratio (d/D) 0.892 0.689 0.813The numbers correspond to P values. Significant values (P < .05) are marked in grey.The histomorphometric data assessment2 used for the actual study included thefollowing parameters:– The cross-sectional area of the nerve– The total fiber number (N)– Fiber density– Fiber diameter (D)– Axon diameter (d)– Myelin thickness (M), and– Axon/fiber ratio or g ratio (g = d/D)Statistical analysisData analysis was performed using version 2.11.0 of R software and its package “stats”to correlate electrophysiological and histomorphological parameters.6 Since the histomor-phological parameters can be considered as independent and the electrophysiological pa-rameters as dependent variables, linear regression analysis has been chosen to correlatethe 2 methods. A normal distribution was expected for all parameters. Linear regressionanalyses have been performed between 1 of the 3 electrophysiological parameters and 1 ofthe 7 different histomorphological parameters mentioned earlier at a time. The algorithmused to fit linear models was the one proposed by Chambers.7 The level of significance afterapplying an F statistic was set by a P < .05. The Institute of Biometry of the Universityof Tuebingen has validated the statistical analysis. The work described in the actual articlefulfils the Uniform Requirements for manuscripts submitted to Biomedical journals.RESULTSThe P values obtained by the F statistic applied to find significant correlations betweenthe histomorphometric and neurographic data after performing a linear regression analysisare shown in Table 2. Significant linear correlations with P < .05 could be found in3 cases. These were between (a) transduction velocity and cross-sectional area of the138MANOLI ET ALnerve (y = −9.96 + 83.42x, P = .009), (b) transduction velocity and total fiber number(y = −15.82 + 83.42x, P = .037), and (c) amplitude and total fiber number (y = –19.43+ 32.6x, P = .026). These 3 linear models are depicted in Figures 1a-c. Concerning thefirst case, an even more significant linear correlation could be observed between the squareroot of the cross-sectional area of the nerve and the transduction velocity (y = –25.71 +73.23x, P = .008). Having in mind the formula for calculation of the circle area, A = πr2,it can be concluded that transduction velocity is linear correlated to the nerve radius ordiameter. Figure 2 is a graphical presentation of this linear fitted model (red line). Dataoriginating from the 2 different studies are presented with different symbols and differentcolors according to their treatment, as described in the legends. Such graphs may be usefultools to compare different treatments or different studies. In this case, we can conclude thatanimals of study B achieved a higher mean transduction velocity than the animals of studyA. This is probably due to the generally slightly larger diameter of the median nerves of therats used for study B with a mean value of 0.217 μm2 than for study A with a mean valueof 0.198 μm2.DISCUSSIONIn this study, a good linear correlation could be obtained between 2 neurographic pa-rameters (transduction velocity and amplitude) and the total fiber number obtained bythe histomorphometric analysis. Moreover, a very good linear correlation was obtainedbetween transduction velocity and nerve diameter. Our results illustrate the progress inthe improvement of histomorphometry and especially of neurography in rodents, as nosignificant correlations between these methods could be obtained in the early phase aftercompletion of functional recovery in several previous studies.8-10 In a previous study, amoderate to good correlation between amplitude and fiber counts demonstrating a diameter3 to 5μm in the peroneal nerve of the rabbit, 4 to 15 weeks after repair, could be obtained.11Dellon and Mackinnon first demonstrated a strong positive correlation between con-duction velocity and fiber diameter as well as between amplitude and number of nerve fibers1 year after nerve repair.12 In our study, no significant correlation between the conductionvelocity and fiber or axon diameter could be obtained. This is probably due to the earlytime point (12 weeks) of neurographic and histomorphometric assessment. Previous studiesevaluating nerve regeneration in the rat across a nerve repair demonstrated that the totalnumber of fibers that reached the distal segment varied with time. In the first few monthsafter surgery, the number of axons will increase dramatically due to axonal sprouting. Someof the axon sprouts make appropriate distal connections and some of them do not, whichresults in a later decrease in fibers. The number of axons reach a plateau between 6 and9 months after repair and return to normal levels within 1 year in the rat model.13,14 Thus,changes in the number of nerve fibers are expected in the first year after nerve repair. More-over, most authors found a decrease in axon diameter concurring with a smaller decreasein fibre diameter up 1.5 years after nerve repair.It has been shown that the ratio of axon to fiber diameter (d/D) or g-ratio remains quiteconstant during regeneration and that conduction velocity depends on a small number of thelargest axons in the nerve.15 However, no significant linear correlation between conductionvelocity and the maximal g-ratio could be found in our data. A significant correlation139ePlasty VOLUME 14between myelin thickness and conduction velocity was expected16,17 but could not beverified in our data. An explanation could be that a significant part of myelinated fibersmay have been sensory fibers that do not have an impact in motor neurography. However,the good correlation between transduction velocity and total fiber number implies that thetotal fiber number may be a better morphological predictor of an effective peripheral nerveregeneration of a mixed nerve than the myelin thickness, especially during the early phaseof regeneration................0.0 0.1 0.2 0.3 0.446810a) y = −9.96 + 83.42xNerve cross sectional area, cm2Transduction velocity, m/s...............................0 2 4 6 8 10 1246810b) y = −15.82 + 3.5xTotal fiber numberTransuction velocity, m/s...............................0 2 4 6 8 10 120100200300400c) y = − 19.43 + 32.6xTotal fiber numberAmplitude (uV)............Figure 1. Fitted linear models (red lines) after regression analysis; between (a) v and cross-sectionalarea of the nerve, (b) v and N, and (c) amplitude and N.CONCLUSIONSInteresting correlations of histomorphometric and neurographic data originating from 2studies using the median nerve rat model could be observed. Our results could validate140MANOLI ET ALmotor neurography as a sensible method for the assessment of regeneration in the mediannerve model of the rat. These findings are also important since histomorphometric andelectrophysiologic measurements enable a more subtle interpretation of peripheral nerveregeneration quality than functional tests do and do not always correlate with nerve sensoryor motor function.18,19 A combined analysis of histomorphometry and motor neurographyenables an even more precise evaluation of the axonal regeneration in the median nervemodel of the rat, making it a powerful model to investigate several conditions that mayinfluence peripheral nerve regeneration, or new reconstruction methods and strategiesbefore applying them on a clinical level.Figure 2. Fitted linear model (red line, y = −25.71 + 73.23x) after re-gression analysis between the square root of the cross-sectional area ofthe nerve and the transduction velocity. Data originating from study Aare depicted by “o” and data originating from study B by “ˆ.” The dif-ferent colors present the different treatments, as described in the legends.Magenta ellipse includes all measurements originating from study A,while green ellipse includes all measurements but one originating fromstudy B.AcknowledgmentsThe authors thank Dr Meisner of the Institute of Biometry of the University of Tuebingenfor validating and supporting the statistical analysis of the data.141ePlasty VOLUME 14REFERENCES1. Bertelli JA, Mira JC. The grasping test: a simple behavioral method for objective quantitative assessmentof peripheral nerve regeneration in the rat. J Neurosci Methods. 1995;58:151-5.2. RaimondoS, FornaroM,Di Scipio F, et al. Chapter 5:methods and protocols in peripheral nerve regenerationexperimental research: part II—morphological techniques. Int Rev Neurobiol. 2009;87:81-103.3. Werdin F, Grussinger H, Jaminet P, et al. An improved electrophysiological method to study peripheralnerve regeneration in rats. J Neurosci Methods. 2009;182:71-7.4. Sinis N, Di Scipio F, Schonle P, et al. Local administration of DFO-loaded lipid particles improves recoveryafter end-to-end reconstruction of rat median nerve. Restor Neurol Neurosci. 2009;27:651-62.5. Sinis N, Manoli T, Schiefer JL, et al. Application of 2 different hemostatic procedures during microsurgicalmedian nerve reconstruction in the rat does not hinder axonal regeneration. Neurosurgery. 2011;68:1399-403, discussion 1403-1394.6. R Development Core Team R: A Language and Environment for Statistical Computing. Vienna, Austria:R Foundation for Statistical Computing; 2010.7. Chambers JM. Chapter 4: linear models. In: Chambers JM, Hastie TJ, eds. Statistical Models in S. PacificGrove, CA: Wadsworth & Brooks/Cole; 1992.8. Munro CA, Szalai JP, Mackinnon SE, Midha R. Lack of association between outcome measures of nerveregeneration. Muscle Nerve. 1998;21:1095-7.9. Kanaya F, Firrell JC, Breidenbach WC. Sciatic function index, nerve conduction tests, muscle contraction,and axon morphometry as indicators of regeneration. Plast Reconstr Surg. 1996;98:1264-71, discussion1272-1264.10. Wolthers M, Moldovan M, Binderup T, Schmalbruch H, Krarup C. Comparative electrophysiological,functional, and histological studies of nerve lesions in rats. Microsurgery. 2005;25:508-19.11. van Neck JW, de Kool BS, Hekking-Weijma JI, Walbeehm ET, Visser GH, Blok JH. Histological validationof ultrasound-guided neurography in early nerve regeneration. Muscle Nerve. 2009;40:967-75.12. Dellon AL, Mackinnon SE. Selection of the appropriate parameter to measure neural regeneration. AnnPlast Surg. 1989;23:197-202.13. Mackinnon SE, Dellon AL, O’Brien JP, et al. Selection of optimal axon ratio for nerve regeneration. AnnPlast Surg. 1989;23:129-34.14. Schaller H-E.. Die Bedeutung des MHC und non MHC fu¨r das allogene periphere Nerventransplantat imTiermodell der Ratte. Habilitationsanschrift Hannover. Hannover, Germany; 1990.15. Gillespie MJ, Stein RB. The relationship between axon diameter, myelin thickness and conduction velocityduring atrophy of mammalian peripheral nerves. Brain Res. 1983;259:41-56.16. McDonald WI. The effects of experimental demyelination on conduction in peripheral nerve: a histologicaland electrophysiological study. II. Electrophysiological observations. Brain. 1963;86:501-24.17. McDonald WI. The effects of experimental demyelination on conduction in peripheral nerve: a histologicaland electrophysiological study. I. Clinical and histological observations. Brain. 1963;86:481-500.18. Martins RS, Siqueira MG, da Silva CF, Plese JP. Correlation between parameters of electrophysiological,histomorphometric and sciatic functional index evaluations after rat sciatic nerve repair.ArqNeuropsiquiatr.2006;64:750-6.19. Vleggeert-Lankamp CL. The role of evaluation methods in the assessment of peripheral nerve regenerationthrough synthetic conduits: a systematic review. Laboratory investigation. J Neurosurg. 2007;107:1168-89.142

Correlation analysis of histomorphometry and motor neurography in the median nerve rat model

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Correlation analysis of histomorphometry and motor neurography in the median nerve rat model

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