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Jun 9, 2005 - motor evoked potentials (MEPs) for monitoring disease ... evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and.
Journal of Neuroscience Research 81:597–604 (2005)

Myelin Deterioration in Twitcher Mice: Motor Evoked Potentials and Magnetic Resonance Imaging as In Vivo Monitoring Tools D. Dolcetta,1* S. Amadio,2 U. Guerrini,3 M.I. Givogri,1 L. Perani,1 F. Galbiati,1 L. Sironi,3 U. Del Carro,2 M.G. Roncarolo,1 and E. Bongarzone1* 1

Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milano, Italy Neurology Department, San Raffaele Scientific Institute, Milano, Italy 3 Department of Pharmacological Sciences, Milano, Italy 2

We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 6 0.49 msec at P27 to 6.45 6 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 6 0.81 msec at P27 to 2.5 6 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals. VC 2005 Wiley-Liss, Inc. Key words: globoid cells leukodystrophy; magnetic resonance; motor evoked potentials; Twitcher mouse

Deficiency of the lysosomal enzyme galactocerebrosidase (GALC; EC 3.2.1.46) leads to globoid cell leukodystrophy, or Krabbe disease (GLD; Wenger et al., 2000). The main neuropathological hallmarks of GLD are the ' 2005 Wiley-Liss, Inc.

progressive accumulation of GALC natural substrates: galactocerebroside (Galc) and the highly toxic lipid galactosil-sphingosine (psychosine), mainly within oligodendrocytes and Schwann cells (Igisu and Suzuki, 1984); this leads to abundant cell death (Im et al., 2001; Tohyama et al., 2001; Jatana et al., 2002), diffuse neuroinflammation, and globoid cell formation (LeVine et al., 1994). These alterations invariably lead to central and peripheral dysmyelination and death within few years after clinical onset (Hagberg, 1984). The Twitcher mouse, a natural occurring GALC-deficient mutant, develops most of the pathological hallmarks seen in human GLD patients, leading to early (P45) death of the mice, and thus represents an ideal animal model of severe forms of human GLD (Kobayashi et al., 1980; Igisu et al., 1983). The first symptom in the Twitcher mouse is the reduced gain in body weight, clearly noticeable as soon as P15. After P20, impairment in muscular strength appears, but hind limb paresis becomes evident only around P30, together with resting tremor (twitching). At about the same age, gain in body weight arrests, and the Twitcher mouse enter in the last phase of the disease. The severe decrease in body weight (likely because of feeding difficulties) and spasticity bring death, at about P42 (Wenger et al., 2000). CNS and peripheral deterioration in Twitcher mice have been studied by evaluation of clinical parameters, such as life span and gain in body weight and by postmortem biochemical and pathological techniques. However, tests for in vivo assessment of CNS compromise of *Correspondence to: Dr. Diego Dolcetta, Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, Milano 20132, Italy. E-mail: [email protected] Dr. Ernesto R. Bongarzone, Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, Milano 20132 Italy. E-mail: [email protected] Received 13 December 2004; Revised 15 March 2005; Accepted 29 April 2005 Published online 9 June 2005 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jrn.20574

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Twitcher mice have been largely missing. In humans, electrophysiological methods represent a fundamental tool for clinical evaluation of myelin maturation after birth (Lang et al., 1985; Garcia-Garcia and Callaja-Fernandez, 2004). In healthy humans, myelin maturation appearing during childhood development of CNS and PNS is paralleled by a progressive decrease of conduction latencies (Lang et al., 1985; Garvey et al., 2003). For mice, motor evoked potentials (MEPs) have recently shown notable sensitivity in recording slowed central conduction latencies (Pluchino et al., 2003; Biffi et al., 2004). Myelination in rodents is characterized by progressive increase of axonal diameter and myelin sheath thickness (Uematsu et al., 1996), together with internode elongation (Bjartmar, 1996) within corticospinal tract fibers during first weeks of life. Thus, the study of conduction velocities of adult Twitcher mice, as recently shown in a mouse model for multiple sclerosis (MS; Pluchino et al., 2003), would likely allow the assessment of nervous system electrical properties at both central and peripheral levels in vivo. In addition to MEPs, brain magnetic resonance imaging (MRI) is a highly sensitive tool for studying CNS myelination in humans as well as for the detection of developmental, clinical, or subclinical white matter abnormalities (Miot-Noirault et al., 1997). Recently, MRI has been validated for studying rodent CNS myelination (Matsumae et al., 2003), offering its potential as a sensitive tool for diagnosis and follow-up of Twitcher mice undergoing correcting therapies. Here we have adapted and optimized MEP and MRI techniques for in vivo assessment of CNS myelination during postnatal development in the Twitcher mouse. Our studies allowed a comprehensive evaluation of the electrical conductivity within corticospinal tracts in living Twitchers, showing a drastic increase in the latency of neurotransmission in the Twitcher CNS and PNS. These findings were correlated with T2-weighted MRI scans showing severe dysmyelination in white matter structures of the mutant brain, such as the corpus callosum. MATERIALS AND METHODS Animals and Anesthetic The original breeder pairs (heterozygote), on the C57BL/6 background, were obtained from Jackson Laboratory (Bar Harbor, ME), and the colony has been maintained in our institution. Genotype was determined by polymerase chain reaction with genomic tail DNA (Sakai et al., 1996). Avertin (2,2,2-tribromoethanol; Sigma, St. Louis, MO), was used to anesthetize mice. We noted that avertin preparations including 2-methyl-2-buthanol (Papaioannou and Fox, 1993) led to altered MEPs recordings. Therefore, we administrated 0.02 ml/g body weight of a 1.25% solution of Avertin without 2-methil-2-buthanol, allowing a profound, safe, but short anesthesia. In total, 19 Twitchers and 18 healthy littermate controls were dedicated to MEPs, and 9 twitcher and 12 littermates were dedicated to MRI studies. Histopathology Mice were sacrificed by CO2 inhalation, perfused with heparin/saline, and fixed with 4% paraformaldehyde. Brains

and spinal cords were processed for paraffin embedding and were sectioned at 10 mm thickness. Samples were stained by using the Bielchowsky method (neuronal fibers) and the Luxol fast blue/PAS (myelin and globoid cells). MRI For MRI evaluations, anesthetized mice were placed on the animal holder into the 4.7-T, vertical 15-cm bore magnet of a Bruker spectrometer (AMX3 with microimaging accessory). A 3.8-cm-diameter birdcage coil was used for the imaging. A three-orthogonal-plane gradient echo scout acted as a geometric reference. Subsequently, six contiguous 1.1-mmthick coronal slices were acquired rostrocaudally starting from the olfactory bulb [then approximately at 2.0, 0.9 mm rostrally to bregma and –0.2, –1.3, –2.4, –3.5 mm caudally to it, in accordance with the Paxinos and Franklin (2001) mouse brain atlas]; by using turbo spin echo T2 weighted (Bruker RARE), with 32 echoes per excitation, 6 msec of interecho time, 98.3 msec equivalent echo time, and 4 sec repetition time, 32 averages were acquired over 8.5 min. Neurophysiological Methods Sciatic nerve motor conduction velocity. Sciatic nerve motor conduction velocity (MCV) was assessed according to previously described techniques (Zielasek et al., 1996). Briefly, compound motor action potential (CMAP) was obtained, stimulating the nerve at the ankle and ischiatic notch with a pair of needle electrodes and recording in distal hind limb muscles. The active electrode was placed in the middle of plantar muscles, whereas the reference was inserted subcutaneously in the second digit. MCV was measured by dividing the distance between the two points of stimulation by the difference between proximal and distal CMAP latencies. F-waves were recorded from proximal nerve segments and motor roots with the same montage as described for MCV (Toyoshima et al., 1986). Transcranial stimulation. Cortical motor evoked potentials (cMEP) were elicited with a pair of needle monopolar electrodes placed over the intact scalp. The intensity of transcranial stimulation was settled at about 50% greater than motor threshold, which is the lowest current intensity for obtaining reliable muscle responses. The position of stimulating electrodes, which generated electric fields oriented through the mouse primary motor area (Chiba et al., 2003), was slightly modified in order to deliver the lowest current intensity to excite motor cortex. Muscle recordings. Muscle responses evoked by descending volleys through transynaptic depolarization of spinal a-motoneuron were recorded with a bipolar montage as described for the MCV method. The electromyographic signal was bandpassed with 60 Hz to 5 kHz filters and recorded with a Myoquick electromyographer (Micromed, Mogliano Veneto, Italy). A ground electrode filled with electroconductive gel was placed at the ankle. To distinguish EMG signal from background noise, responses lower than 50 mV were excluded. Because of variability in cortical responses, 8 to 10 evoked responses were considered. The onset of the first, usually negative, deflection was taken as MEP latency.

In Vivo Evaluation of Twitcher Mice

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Fig. 1. Histopathology of Twitcher brain. A–H: Luxol fast blue-PAS staining of corpus callosum (A–F,H) at different ages and of anterior commissure (G). Globoid cells were detected at P30 (G,H, arrows) and P40 (C, arrows). Signs of dysmyelination were evident at P45 (E). I–L: Axonal degeneration in fibers within the corpus callosum (I) and vacuolation in corticospinal tract fibers (K; Bielchowsky staining) were detected only after Twitcher mice reached P40. A–D,I,J: 20; E–H,K,L: 40.

Central conduction time. Central conduction time (CCT), an index of the propagation time of stimulus-related volleys descending along corticospinal tracts, was calculated as the difference between the latency of the spinal motor evoked potential (sMEP), i.e., latency of muscle responses to stimulate lumbar motor roots electrically minus that of the cortical MEP (cMEP).

RESULTS Dysmyelination, Infiltration of Globoid Cells, and Axonal Degeneration in Late Stages of Twitcher Disease Twitcher mice undergo progressive dysmyelination of the main white matter areas, with profuse infiltration of activated microglia with altered features (globoid cells; Kanazawa et al., 2000). To correlate our MRI and MEPs studies with the course of the neuropathological alterations, we collected brains from Twitcher and healthy wild-type littermates from 30 to 45 days after birth and processed them for Luxol fast blue-PAS and Bielchowsky stainings to evaluate myelin and axonal degeneration. Figure 1A–F shows a progressive deterio-

ration in myelination of the corpus callosum in Twitcher mice. For early stages (P30), we could not detect gross differences between Twitcher (Fig. 1A) and wild-type (Fig. 1B) corpus callosum. However, the Twitcher brain already showed the presence of abundant globoid cells (Fig. 1G,H), accumulated particularly near the ventricular system (Fig. 1H, arrows). At later stages (P40 and particularly P45, the longest survival time of Twitcher mice collected in this study), the Twitcher corpus callosum showed infiltration of globoid cells (Fig. 1C, arrows) and then a severe reduction in size, accompanied by a significant loss of myelin (Fig. 1E). Healthy controls did not contain any globoid cell (Fig. 1D,F). Figure 1G shows the presence of globoid cells (arrows) within an area of robust dysmyelination in the anterior commissure of a Twitcher animal. Although axonal integrity was apparently normal at P30 in Twitcher mice (data not shown), at P40 vacuolation was abundant more than that in age-matched controls within bundles of axons. Vacuolation was detected in commissural axons within the Twitcher corpus callosum (Fig. 1I, arrows) and in axonal bundles

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within the caudate putamen (Fig. 1K, arrows). This is likely related to Galc deposits rather than late axonal degeneration. T2-MRI Detects Lack of Myelin Maturation in Twitcher Brains We evaluated myelin formation in wild-type, heterozygous, and Twitcher mice by T2-weighted MRI at two developmental time points, P32 and P37. At P32, the corpus callosum T2 signal was still uncertain in control wild-type and heterozygote mice, and it was not possible to distinguish it from Twitcher images. Five days later, at P37, control mice showed clear hypointensity of the frontal portion of the corpus callosum (Fig. 2D,F, arrows), less well defined hypointensity associated with cerebral peduncle, internal capsule and fimbria (Fig. 2H,J, arrows), and the caudal portion of external capsule (Fig. 2L, arrows). In contrast, Twitcher mice revealed T2-weighted isointensity of the corpus callosum and of all myelinated structures mentioned above (Fig. 2C,E,G,I,K). Surprisingly, the external capsule signal was not completely absent in Twitcher mice. No difference was seen between wild-type and heterozygote controls (data not shown). Twitcher Mice Show Significant Reduction in Neural Impulse Central Conduction Age-related cortical responses in normal and Twitcher mice were recorded at P20–P22, at P27–P29, and at P30–P32. We were able to record MEPs in healthy controls between P20 and P22, with sMEP latency of 2.55 6 0.35 msec, cMEP latency of 5.75 6 0.64 msec, and CCT of 3.45 6 0.63 msec. However, MEPs in age-matched Twitchers could not be obtained because of the reduced dimensions of the mutant at those postnatal days (Igisu et al., 1983). After P27, Twitcher mice acquired a body size that was compatible with our experimental settings for electrophysiology. At P27, control mice had a CCT of 3.01 msec 6 0.81. At P32, CCT were reduced to 2.5 msec 6 0.27 (Fig. 3). Twitcher mice instead progressively increased their CCT, passing from 5.12 msec 6 0.49 at P27 to 6.45 msec 6 1.96 at P32 (Fig. 3). Figure 4 shows an example of MEP recordings for a control mouse (left column) and an aged-matched Twitcher (right column), clearly indicating increased latency both centrally and peripherally at P32. Table I summarizes the statistical analysis of all electrophysiological parameters obtained at P27 and P32. We found significant altered values in Twitcher mice with prolonged latencies and shortening of motor conduction velocity. At P33 and P34, cMEPs started to be difficult to measure. Twitcher mice older than P35 did not show readable cMEPs. This likely was due to conduction block, in that sMEPs were always still recordable. Therefore, the optimal time frame for MEP recordings in Twitcher mice is between P27 and P32, with con-

Fig. 2. MRI scanning identified areas of dysmyelination in the Twitcher brain. T2-weighted 4.7-T MRI of Twitcher (left) and healthy littermates (right) was performed at P37. Six contiguous 1.1-mm-thick coronal slices were acquired, then approximately at 2.0, 0.9 mm rostrally to Bregma and –0.2, –1.3, –2.4, and –3.5 mm caudally to it, with little discordance resulting from slight differences in brain size. Whereas in healthy littermates MRI shows a defined hypointense signal in the anterior part of corpus callosum (CC; D,E, arrows), this signal is completely absent in the Twitcher brain (C,E, arrows). Other myelinated structures, such as internal capsule (ic), cerebral peduncle (cp), and fimbria (fi), are absent in Twitcher (G,I, arrows) but present in healthy littermates (H,J, arrows). External capsule (ec) was evident in healthy littermates (L, arrows) and less intense in Twitchers (K, arrows).

sistent and reproducible readings in more than 90% of the mutant mice studied. No statistical difference was obtained in MEPs from wild-type and heterozygote controls (data not shown).

In Vivo Evaluation of Twitcher Mice

Fig. 3. Delayed central conduction time in aging Twitchers. Control mice progressively had shortened central conduction times (CCT) between P27 and P32. In contrast, Twitcher mice showed a significant increase in CCT between those days.

DISCUSSION In this study, we have validated the use of MRI and MEPs as tools for in vivo monitoring of disease progression within the CNS of the Twitcher mouse, the murine model for human GLD. Our work aids in overcoming one major limitation in handling this murine model: the lack of in vivo CNS monitoring tools. MEPs have already been successfully exploited in rats (Fujiki et al., 2004), where magnetic cortical stimulation has also been possible, but much less has been published on MEPs in mouse models, where motor potentials are to be evoked by electrical cortical stimulation (Pluchino et al., 2003). On the other hand, resonance studies on Twitcher have focused only on myelination in optic and trigeminal nerves (Ono et al., 1995). Until now, life span, gain in body weight, and peripheral motor conduction velocity (MCV) have been used for in vivo analysis (Hoogerbrugge et al., 1988), in combination with neuropathological (mainly based on GCs count on PAS-stained slices) and biochemical analyses in post-mortem quantitative studies. The reduced availability of reproducible and sensitive in vivo tests has limited the ability to follow Twitcher mice under different gene and cell therapy treatments. When viewed with the 4.7-T MRI apparatus, the corpus callosum—the most voluminous CNS myelinated structure within the normal brain—appeared as a hypointense signal in T2-weighted sequences as early as P37 in both healthy wild-type and heterozygous littermates. By contrast, age-matched homozygous Twitcher mice revealed T2-weighted isointense signal of the corpus callosum, indicating hypo-/dysmyelination. When examined by histopathological analysis, Twitcher mice showed less intense myelin staining later (around P40), accompanied by detection of abundant globoid cells. MR signal, in T2 sequences, depends on abundance of free water molecules.

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MR corpus callosum physiological hypointensity, then, depends on their reduction, because of myelin compaction and decrease of water-lipid ratio. Its hypointensity is then proportional to myelin maturation. This is already evident in healthy mice (heterozygous or wild-type littermates) at about P33 but is constantly present and with definitive intensity at P37. Luxol fast blue reagent reacts with phospholipids present in normal myelin. In myelin degeneration processes, phospholipids are progressively transformed into hydrophobic lysophospholipids, which do not react with Luxol fast blue (Mazzi, 1977). By the Luxol fast blue method, degenerated myelin is then revealed by a blue staining lighter than expected (see Fig. 1G). Luxol blue staining hypointensity is therefore a good marker of myelin degeneration. Thus, the two diagnostic methods, MRI and histology, allow the evaluation of two aspects of the disease: lack of myelin maturation (dysmyelination) and myelin degeneration (demyelination), the latter revealed by Luxol staining only in the very advanced phase of the Twitcher clinical course. Our study demonstrates that in vivo MRI neuroimaging performed at about P37 allows anticipated evaluation of myelination in the Twitcher brain and could be a useful assessment tool for myelin study in experimental therapeutic protocols. We next studied the functionality of corticospinal tracts by recording MEPs. Healthy mice physiologically improved central motor conduction velocity in adult age. They reached definitive values by the second month of life, passing from nearly 3.45 6 0.63 msec, at P21—the earliest age at which MEPs were recordable—to 2.5 6 0.27 msec, at P32 and older, likely through sustained myelin maturation (Hildebrand and Waxman, 1984; Bjartmar, 1996; Hardy et al., 1996). The difference in MEPs values between controls and Twitcher mice rapidly increased after P21, in parallel with MCV impairment. Twitcher mice never displayed CCT below 4.45 msec at the different times tested. At P32, Twitchers showed twofold increases in CCT values. The capacity of MEP measurements to detect functional impairment of corticospinal pathways was not unexpected. Multiple volleys produced by motor cortex stimulation have to cover the stretch of corticospinal tracts between brain and spinal cord before synapsing with spinal a-motoneurons at the lumbar level. The longer the tract explored, the greater the chance that it might be injured. Because the disease severely affects spinal cord (Taniike and Suzuki, 1994), it is conceivable that, after P35, the level of myelin injury exceeds the chances of nervous impulses covering the whole tract and reaching the muscle. Insofar as GLD involves both CNS and PNS, the lack of excitability of cortical responses recorded from muscle, as long as peripheral conduction is ensured, must be explained as the result of severe damage mainly involving corticospinal system. cMEP are generated by trains of descending volleys converging on spinal a-motoneurons. Dispersion of conduction velocity values, resulting from multifocal demyelination and partial conduction blocks, might prevent single volleys from synchronizing and

Figure 4.

In Vivo Evaluation of Twitcher Mice TABLE I. Neurophysiological Readings* Controls (n ¼ 19) MCV (m/sec) P27–32 CMAPDML P27–32 (msec) CMAPampl (mV) f-Wavelat (msec) P27–32 sMEPlat (msec) P27–29 sMEPlat (msec) P30–32 cMEPlat (msec) P27–29 cMEPlat (msec) P30–32 cMEPampl (mV) CCT (msec) P29–29

26.84 1.1 10.19 5.35 2.2 2.45 5.2 5.37 0.4 3.01

6 6 6 6 6 6 6 6 6 6

4.82 0.21 6.65 0.7 0.17 0.31 0.98 0.71 0.29 0.81

Twitcher (n ¼ 18) 7.0 2.18 1.7 13.18 5.42 6.15 10.52 12.51 0.1 5.12

6 6 6 6 6 6 6 6 6 6

1.31 0.67 0.6 3.79 0.48 1.02 0.79 2.77 0.06 0.49

*MCV, ischiatic nerve motor conduction velocity; CMAP, compound motor action potential; DML, distal motor latency; ampl, amplitude; lat, latency; cMEP, cortical motor evoked potential; sMEP, spinal motor evoked potential; CCT, central conduction time. Values were verified with twotailed t-test for unpaired data and were found all to be statistically significant, with P < 0.001, except for cMEP voltage, significant with P < 0.002.

reaching the threshold for motoneuron discharge (Day et al., 1987). Early after birth, GALC activity is needed for completing both CNS and PNS myelination (Coetzee et al., 1996; Marcus and Popko, 2002). Thus, its absence or impairment—as occurs in Twitcher mice—severely compromises normal compaction of myelin sheets into myelinated structures such as corpus callosum and corticospinal tracts. Galactocerebroside and particularly psychosine neurotoxicity and subsequent dysmyelination likely lead to the progressive functional impairment (e.g., early disappearance of cMEP) found in this study. Our results have identified a window of time during which pathological MEPs (P27–P32) and MR images (after P37) are recordable, showing differences from wild-type littermates. Both methods are performed in vivo, are absolutely well tolerated, and allow follow-up for animals subjected to therapeutic experimentation. In our case, because of the extremely short life span of untreated Twitcher, longitudinal evaluation (beyond 5 weeks of age) of these mice by MEPs could not be performed. These findings seem to run in parallel more with the clinical course of Twitcher mice than with traditional histopathology. The strong delay in central conduction, the impossibility of recording it after P35, and the complete absence of the T2-MRI corpus callosum hypointensity at b

Fig. 4. Motor evoked potential readings. Left column: CMAP obtained by stimulation of ischiatic nerve at the ankle (trace 1) and at the ischiatic notch (trace 2) in a healthy P30 mouse. Trace 3 shows the motor evoked potential obtained by stimulation of lumbar motor roots (sMEP). Traces 4 show cortical motor evoked potential (cMEP) following 10 trials of transcranial stimulation. The same traces are superimposed (trace 5) and averaged (trace 6). The average of the previous 10 traces was used for measuring central conduction time (CCT), as time difference between cMEP and sMEP latencies. Right column traces represent typical recordings from an age-matched Twitcher mouse. Both peripheral and central responses are severely delayed, low in voltage, and dispersed in shape. Note the different sensitivity between Twitcher recordings and control, while the same time scale was maintained.

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P37 appear more in line with the severity of the disease in the late stage in the life of Twitcher mutants. In summary, traditional morphological analysis is a sensitive tool for detecting significant myelin degeneration during the terminal phase of the disease, whereas MRI detects the lack of physiological myelin compaction. MEPs allow evaluation of both brain and spinal cord conduction. Experimental gene and cell therapy approaches in mouse models for human lysosomal storage diseases such as GLD aiming at CNS neuroprotection would clearly benefit from reliable in vivo CNS assessment tools such as those described here. ACKNOWLEDGMENTS This work was funded by a Telethon Program Project Grant to E.B. We thank Drs. Stefano Pluchino and Giorgio Giaccone for valuable advice. REFERENCES Biffi A, De Palma M, Quattrini A, Del Carro U, Amadio S, Visigalli I, Sessa M, Fasano S, Brambilla R, Marchesini S, Bordignon C, Naldini L. 2004. Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells. J Clin Invest 113:1118–1129. Bjartmar C. 1996. Oligodendroglial sheath lengths in developing rat ventral funiculus and corpus callosum. Neurosci Lett 216:85–88. Chiba S, Iwasaki Y, Sekino H, Suzuki N. 2003. Transplantation of motoneuron-enriched neural cells derived from mouse embryonic stem cells improves motor function of hemiplegic mice. Cell Transplant 12:457–468. Coetzee T, Fujita N, Dupree J, Shi R, Blight A, Suzuki K, Suzuki K, Popko B. 1996. Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability. Cell 86:209–219. Day BL, Rothwell JC, Thompson PD, Dick JP, Cowan JM, Berardelli A, Marsden CD. 1987. Motor cortex stimulation in intact man. 2. Multiple descending volleys. Brain 110:1191–1209. Fujiki M, Kobayashi H, Inoue R, Ishii K. 2004. Immediate plasticity in the motor pathways after spinal cord hemisection: implications for transcranial magnetic motor-evoked potentials. Exp Neurol 187:468–477. Garcia-Garcia A, Calleja-Fernandez J. 2004. [Neurophysiology of the development and maturation of the peripheral nervous system]. Rev Neurol 38:79–83. Garvey MA, Ziemann U, Bartko JJ, Denckla MB, Barker CA, Wassermann EM. 2003. Cortical correlates of neuromotor development in healthy children. Clin Neurophysiol 114:1662–1670. Hagberg B. 1984. Krabbe’s disease: clinical presentation of neurological variants. Neuropediatrics 15(Suppl):11–15. Hardy RJ, Friedrich VL Jr. 1996. Progressive remodeling of the oligodendrocyte process arbor during myelinogenesis. Dev Neurosci 18:243–254. Hildebrand C, Waxman SG. 1984. Postnatal differentiation of rat optic nerve fibers: electron microscopic observations on the development of nodes of Ranvier and axoglial relations. J Comp Neurol 224:25–37. Hoogerbrugge PM, Suzuki K, Suzuki K, Poorthuis BJ, Kobayashi T, Wagemaker G, van Bekkum DW. 1988. Donor-derived cells in the central nervous system of twitcher mice after bone marrow transplantation. Science 239:1035–1038. Igisu H, Suzuki K. 1984. Progressive accumulation of toxic metabolite in a genetic leukodystrophy. Science 224:753–755. Igisu H, Shimomura K, Kishimoto Y, Suzuki K. 1983. Lipids of developing brain of twitcher mouse. An authentic murine model of human Krabbe disease. Brain 106:405–417. Im DS, Heise CE, Nguyen T, O’Dowd BF, Lynch KR. 2001. Identification of a molecular target of psychosine and its role in globoid cell formation. J Cell Biol 153:429–434.

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