Spontaneous long-term remyelination after traumatic spinal cord injury in rats

Ž .Brain Research 782 1998 126–135

Research report

Spontaneous long-term remyelination after traumatic spinal cord injury inrats

Hermelinda Salgado-Ceballos a,b,), Gabriel Guizar-Sahagun a,b, Alfredo Feria-Velasco c,Israel Grijalva a,b, Laura Espitia b, Antonio Ibarra a,b, Ignacio Madrazo b

a Unit of Medical Research in Neurological Diseases, Instituto Mexicano del Seguro Social, Mexico, DF, C.P. 06720, Mexico´ ´b Proyecto Camina A.C., Mexico DF, C.P. 14050, Mexico´ ´

c DiÕision of Experimental Pathology and Biotechnology, CIATEJ, Guadalajara, Jalisco, Mexico´

Accepted 14 October 1997

Abstract

The capability of the central nervous system to remyelinate axons after a lesion has been well documented, even though it had beenŽ .described as an abortive and incomplete process. At present there are no long- term morphometric studies to assess the spinal cord SC

remyelinative capability. With the purpose to understand this phenomenon better, the SC of seven lesionless rats and the SC of 21 ratssubjected to a severe weight-drop contusion injury were evaluated at 1, 2, 4, 6, and 12 months after injury. The axonal diameter and the

Ž .myelination index MIsaxolemmal perimeter divided by myelinated fiber perimeter were registered in the outer rim of the cord at T9SC level using a transmission electron microscope and a digitizing computer system. The average myelinated fiber loss was 95.1%. Onemonth after the SC, 64% of the surviving fibers were demyelinated while 12 months later, only 30% of the fibers had no myelin sheath.

Ž .The MI in the control group was 0.72"0.07 X"S.D. . In the experimental groups, the greatest demyelination was observed twoŽ . Ž .months after the lesion MIs0.90"0.03 , while the greatest myelination was observed 12 months after the injury MIs0.83"0.02 .

Ž .There was a statistical difference p-0.02 in MI between 2 and 12 months which means that remyelination had taken place.Ž .Remyelination was mainly achieved because of Schwann cells. The proportion of small fibers diameters0.5 mm or less considered as

axon collaterals, increased from 18.45% at 1 month to 27.66% a year after the contusion. Results suggest that remyelination is not anabortive phenomenon but in fact a slow process occurring parallel to other tissue plastic phenomena, such as the emission of axoncollaterals. q 1998 Elsevier Science B.V.

Keywords: Demyelination; Myelination index; Schwann cell; Neural plasticity; Spinal cord contusion; Paraplegia

1. Introduction

Ž .Mechanical trauma to the spinal cord SC producesaxonal destruction and extensive demyelination by damag-ing, not only axons, but also oligodendrocytes and astro-

w xcytes 8,9,26 . As a result, axons that survive the injurylose their myelin sheath and cannot be remyelinated, whileaxons or collaterals, resulting from the plastic mechanisms

w xof the SC, are not myelinated 8,9,21,61 .Myelin is essential for normal nervous system function

w x57 so, loss of the axon myelin sheath has importantrepercussions on nerve physiology as may be a decrease inconduction velocity or overt nerve conduction block thatmay play a major role in the neurological deficits after SC

) Corresponding author. Calzada de Tlalpan 4430, Colonia TorielloGuerra C.P. 14050, Mexico DF, Mexico. Fax: q52-5-5-73-55-45.´ ´

w xinjury 61 . As a consequence, functional recovery of thispathology depends at least in part on nervous system

w xremyelinating ability 40,44,60,61 .Research over the past several decades has demon-

strated that myelin repair in the central nervous systemŽ .CNS is a normal physiological response to myelin injuryw x13,38,49 . However, some authors regard remyelination inthis system as an abortive and incomplete phenomenonw x3,5,6,11,18,25,31–33,39,47,48,56 .

The availability of numerous models of CNS demyeli-nation amenable to experimental manipulationw x20,30,37,41,45,54,62 has allowed a broad range of ap-proaches to develop and for evaluating therapies designed

w xto promote CNS remyelination 19,42–45,55 . However,with the aim to design rational therapeutic strategies indifferent pathologic processes related to SC injuries, it isnecessary to clearly understand the spontaneous plastic

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 01252-3

( )H. Salgado-Ceballos et al.rBrain Research 782 1998 126–135 127

mechanisms subjacent in each one of those processes andnot to negatively interfere with them.

The long-term morphometric effect on the axon diame-ters and axonal myelination of the adult rat SC fibers afteran experimental weight-drop contusion injury was ana-lyzed in order to better understand the CNS remyelinativecapacity and knowing whether remyelination constitutes anabortive and incomplete process in the biology of the SC.

2. Materials and methods

2.1. Surgery and post-surgical care

Twenty-eight Long-Evans female rats 14 to 16 weeks ofage, weighing 240 to 260 g, were anesthetized with an

Ž . Ž .intramuscular IM mixture of ketamine 77.5 mgrkg bwŽ .and xylacine 12.5 mgrkg bw . Benzatin penicillin

Ž .240,000 units was administered IM, as a single dose,once the rat was anesthetized.

The protocol was accepted by the hospital’s EthicsCommittee while considering the proper handling, hous-ing, surgical and postsurgical care of the animals. Underaseptic conditions, each rat was subjected to a one-level

Ž .laminectomy T9 , exposing the dorsal surface of the SC,without disturbing the meninges. Under stereotactic con-

Ž .trol, a severe spinal cord contusion SCC was causedw xusing the weight-drop method described by Allen 1 and

w xmodified by our group 28 . For a brief moment, a 15 gstainless steel cylinder with a flat 2 mm diameter tip, wasdropped from a height of 10 cm, through a guide tube ontothe exposed dura mater. The formation of a hematoma inthe impacted area was corroborated and, finally, the mus-cle aponeurosis and the skin were separately sutured overthe laminectomy site with 5-0 nylon.

After the surgical procedure, the rats were placed in aŽcritical care unit for small animals Schroer Manufactur-

.ing, Kansas City, KA until their recovery from the anes-thesia. The rats were fed ad libitum and caged separately.Neurogenic bladder and intestine were manually managedtwice a day until automatism was recovered.

2.2. Experimental groups

The time course of the axonal diameter and myelinsheath morphometric alterations were recorded in 21 rats,

Ž .divided in 5 different subgroups studied at 1 ns4 , 2Ž . Ž . Ž . Ž .ns5 , 4 ns5 , 6 ns4 and 12 ns3 months afterthe severe SCC. As a control group, seven animals werestudied without lesion of the SC.

2.3. Electron microscopy

Rats were anesthetized as described above and perfusedintra-aortically at 30 mlrmin and at room temperature

Žwith 100 ml of 0.15 molrl sodium cacodylate buffer pH.7.4 plus 1000 units of heparin, followed by 500 ml of a

fixative solution containing 2.5% glutaraldehyde, 2% para-formaldehyde, 0.05% CaCl and 0.025 molrl sucrose2

Ž .solution in 0.1 molrl cacodylate buffer pH 7.4 . Afterperfusion, the SC corresponding from T8 to T10 levelswas dissected, removed and sectioned transversely at the

Žepicenter zone highest tissue destruction transverse area.of the SC . The blocks obtained were placed in the same

fixative used for perfusion for 2 h and then in 0.1 molrlcacodylate buffer for 14 to 16 h. The blocks of approxi-mately 1 mm3 were post-fixed for an hour with aqueous0.1% osmium tetroxide solution. They were then dehy-drated in graded ethanol, and embedded in EPON 812resin. Semithin coronal sections 1 mm thick were stainedwith toluidine blue and observed under a light microscopein order to identify the area corresponding to the peripheralsubpial rim of white matter with preserved axons at theepicenter injured zone. The material was resectioned usingan ultramicrotome, then sections of approximately 70 nmthick were obtained which were collected on copper grids,contrasted with uranyl acetate and lead citrate and ob-served under a Zeiss EM-10 transmission electron micro-scope. The negatives were obtained at =3150 and 6300 ona 35 mm format.

2.4. Image analysis

The negatives were projected on a digitizing tabletŽ .Hewlett Packard 9874A connected to a computer systemŽ .Hewlett Packard 9825B . Digitization of fibers was madetracing their perimeters with an electronic pen. Repetitivemeasurements obtained by digitizing the same fibers weredone achieving a variation of 0.38% between two differentinvestigators and 0.32% by the same investigator. Theaxon diameters were calculated from perimeter measure-ments assuming the perimeter to be that of a perfect circleto avoid errors due to fixation changes in axon shape. Inorder to obtain the relative thickness of the myelin sheathsŽ . Ž .myelination index or MI , the inner axon and outerŽ .axonqmyelin perimeters of each fiber were calculatedand then the inner perimeter was divided by the outerperimeter.

All data were stored in a AcerMate DX4 computer andanalyzed using the Lotus 1-2-3 program. The mean MI ofeach group was obtained. If the inner perimeter divided bythe outer perimeter representing the MI was equal to 1, theaxons had no myelin and the farther away from 1, thethicker the myelin sheath was.

2.5. Statistical analysis

Data were analyzed by the Number Cruncher StatisticalSystem. The sesgo and kurtosis did not have a normaldistribution. Therefore the nonparametric Mann–WhitneyU-test was used with the aim to compare the control MIwith the corresponding MI in the injured groups. TheSpearman’s Rho test was used to analyze the relationship

( )H. Salgado-Ceballos et al.rBrain Research 782 1998 126–135128

Fig. 1. Caliber distribution of axons at the T9 cord segment of control and injured rats. The data obtained from all injured groups were pooled to generatethe ‘injury’ group data. Injured rats had a selective loss of large diameter myelinated axons as shown by the shift in the caliber spectrum to the left.

between axon diameters and the time course after contu-sion. We used descriptive statistics too.

3. Results

3.1. Number of axons and axon diameter

One month after the SCC, the average loss of myeli-nated fibers studied in this experimental severe lesionmodel was 95.1%. In other words, only 4.9% of the fivers

survived. Twelve months after the lesion, the populationgrew somewhat to 6.3%.

In the control group the axon diameter distribution wasunimodal. Small-diameter axons, 0.5 mm or less, repre-

Ž .sented a minority of 1.08%. There were few axons 3.5%with 5.1 mm or more in diameter. Many caliber axonsŽ .31.57% were between 2.1 and 5 mm and most axon

Ž . Ž .diameters 63.85% were between 0.6 and 2 mm Fig. 1 .In the injured group, where the data of all SCC groups

were pooled in order to generate this set of data the axondiameter, distribution was bimodal. There was a significant

Table 1Axonal diameter distribution by group and their correlation coefficients

Ž . Ž .Axon diameter mm AD distribution % by month after SCC Correlation coefficientŽ .Spearman’s RhoControl 1 2 4 6 12

)0.5 or less 1.08 18.35 20.17 24.87 19.53 27.66 0.830.6–1.0 21.35 36.75 38.53 32.34 31.30 40.72 0.491.1–1.5 25.02 17.53 13.43 13.59 12.96 17.46 y0.60 P

1.6–2.0 17.48 9.33 8.94 8.60 9.93 6.56 y0.66 P

2.1–2.5 11.80 5.69 7.47 6.90 7.80 2.81 y0.492.6–3.0 9.21 4.56 4.49 4.53 6.10 2.20 y0.60 P

3.1–3.5 4.65 2.97 2.89 2.88 5.06 1.27 y0.433.6–4.0 2.97 1.85 2.11 2.06 2.03 0.77 y0.66 P

4.1–4.5 1.81 1.18 0.77 1.29 2.27 0.33 y0.264.6–5.0 1.13 0.56 0.34 0.93 1.27 0.11 y0.235.1 or more 3.50 1.23 0.86 2.00 1.75 0.11 y0.54 P

Ž . Ž . Ž . Ž . Ž . Ž .ns7 ns4 ns5 ns5 ns4 ns3

Ž .Axonal diameter AD distribution showed an important increase on small axons and a decrease on the big axons.Ž) . Ž .A high positive Spearman’s Rho correlation coefficient , means that while more time elapsed after a severe spinal cord contusion SCC , there were

Ž .more small axons 0.5 mm or less .Ž .A moderate negative correlation coefficient P , means that while more time elapsed after the lesion, less medium caliber axons were seen.

The rest of the correlations were low and always negative.The number of animals per group was referred to as n.


Table 2Percentage of axons surviving after a spinal cord contusion and the corresponding percentage of myelinated and nonmyelinated axons

Groups % of axons surviving % of total population % of myelinated population % of nonmyelinated population

Axons Sproutings Axons Sproutings Total Axons Sproutings Total

Control 100.0 98.9 1.1 98.6 0.4 99.0 0.3 0.7 1.01 month 4.9 81.6 18.4 43.2 0.9 44.1 38.3 17.6 55.92 months 5.3 79.8 20.2 34.3 1.6 35.9 45.5 18.6 64.14 months 5.3 75.2 24.8 42.7 4.6 47.3 32.5 20.2 52.76 months 5.8 80.5 19.5 51.7 0.2 51.9 28.8 19.3 48.11 year 6.3 72.3 27.7 62.1 7.8 69.9 10.3 19.8 30.1

Percentage of axons surviving after spinal cord contusion derived from the total axon population in the control group in the area corresponding a peripheralring of white matter from the spinal cord, and the fibers surviving the lesion at this area in the experimental groups. Axons were identified when fiberdiameters )0.5 mm.Axon collaterals were identified when fiber diameterss or -0.5 mm.

increase in the proportion of the small caliber axons withan important loss of the largest. Small-diameter axons 0.5mm or less represented 22.12%, while 1.1 mm or morediameter axons diminished in different proportions withrespect to the control group even though the majority of

Ž . Žcaliber axons 59.7% were between 0.6 and 2 mm Fig..1 .

Ž .The proportion of small fibers 0.5 mm or less in-creased from 1.08% in the control group, to 27.66% at 1 yrafter the SCC. These fibers were considered as axoncollaterals or sproutings. There was a high correlationŽ .Spearman’s Rho correlation coefficients0.83 betweenthe time course after the SCC and the increase of thesesmall axons. The increase on 0.6 to 1 mm caliber axons

Fig. 2. Cross-sectional electron micrograph 2 months after a severe spinal cord contusion. The area corresponds to a peripheral subpial rim of white matter.Ž .There are a lot of small unmyelinated axons arrowheads invested either by Schwann cells or by astrocitic processes and some Schwann cells myelinated

Ž .axons arrows . Scale bar, 1.5 mm.


from 21.35% in the control group to 40.72% at 12 months,Žwas moderately significant Spearman’s Rho correlation

.coefficients0.49 . Axons with 1.1 mm or more decreasedŽin the injured groups Spearman’s Rho correlation coeffi-

.cient between y0.54 to y0.66 and showed a goodŽ .negative correlation Table 1 .

3.2. Myelin

Two months after the SCC, about 36% of the fiberswere myelinated and 64% had no myelin sheath. This is incontrast with the observations over 12 months when 70%of the fibers were myelinated and only 30% nonmyelinatedŽ .Table 2 .

One and two months after the lesion, several demyeli-nated axons were seen through the lesion site, eitherisolated or in small clusters sheathed by Schwann cell

Ž .cytoplasm or astrocyte processes Fig. 2 . By 4 and 6months, there were fibers with partial remyelination andnumerous axons were surrounded by a thin myelin sheath

Ž .contrasting with those with thick myelin sheaths Fig. 3 .These fibers were latter identified by electron microscopyas remyelinated fibers by Schwann cells. Thus, at 12months after the SCC, most axons of various diameters

had thick myelin sheaths characteristic of those producedŽ .by Schwann cells Fig. 4 and most small, nonmyelinated

axons were sheathed by the cytoplasm of those cells or byastrocytic processes.

In this paper, the ratio of Schwann cell or oligodendro-cyte myelinated axons was about 8:1, 1 month after theSCC while at 12 months, it was 23:1 in one case. In theother two cases myelination was achieved only by Schwanncells.

Large axons were myelinated by Schwann cells in a 1:1ratio, while medium caliber axons were frequently, but notalways, isolated in a separate trough of a Schwann cell,even though the same cell unsheathed other nonmyelinatedaxons and smaller axons were unsheathed together by oneSchwann cell cytoplasmic process.

Schwann cell myelinated axons were identified basedon the fact the Schwann cells produce thicker, more com-pact myelin sheaths than those produced by oligodendro-cytes. The presence of Schwann cell nuclei and theircytoplasm sheathing the axon, the identification of a cyto-plasm ring in the periphery of the Schwann cell and theaxon it is myelinating, and the displacement of the CNS

w xglia 11,46 . On the other hand, oligodendrocyte myeli-nated axons were identified by a typically ‘puffing’ of the

ŽFig. 3. Electron micrograph of the injury site 6 months after the lesion that shows some thin CNS-type myelin sheaths relative to axonal diameter large. Ž . Ž .arrows and some astrocytic processes arrowheads surrounding naked axons small arrows . Scale bar, 1.2 mm.


Ž .Fig. 4. The electron micrograph shows remyelinated axons 12 months after the spinal cord contusion. There are axons with thick myelin sheaths asterisksŽ .completely surrounded by a cell disclosing the characteristic morphology of Schwann cell resting on a basal lamina arrowheads . Note the 1:1 axon:

Schwann cell relationship. Scale bar, 0.8 mm.

lamellae at intervals around its circumference and along itslength, giving it a lighter, irregular appearance by compari-

w xson with the Schwann cell myelin 11 .

3.3. Myelination index

Ž .Small fibers 0.5 mm or less could correspond to axoncollaterals. The MI of axon collaterals was studied sepa-

rately from other fibers since these are usually nonmyeli-nated. At 2 months, when demyelination was at its peak,

Ž .the axon MI was 0.90"0.03 X"S.D. and the axoncollaterals MI was 0.98"0.02 while at 12 months, theaxon rate was 0.83"0.02 and the collateral rate was

Ž .0.90"0.04 Table 3 .In the control group, the MI had an unimodal distribu-

tion. Nearly 80% of the 0.6 mm or more diameter axons

Table 3Myelination index time course distribution on axons and collateral axons

ŽMonths after spinal cord contusion Axon MI Standard deviation Collaterals MI 0.5 mm Standard deviation.or less diameter axons

Control 0.72 0.07 0.59 0.041 0.86 0.05 0.94 0.052 0.90 0.03 0.98 0.024 0.88 0.06 0.98 0.026 0.87 0.03 0.97 0.0212 0.83 0.02 0.90 0.04

Ž . Ž w x w x.Myelination index MI normal parameters are between 0.60 to 0.80 Goldman and Albus 27 ; Willams and Chalupa 58 .For the control group, the MI was 0.72.The greatest demyelination was observed at 2 months after the SSC and the greatest remyelination was reached at 12 months after the SCC.

Ž .There was a significant difference using the Mann–Whitney U-test ps0.02 between the 2 and 12 month groups.


Fig. 5. A comparative description shows that the control MI strongly contrasts with the MI at 2 months after the spinal cord contusion when demyelinationŽ .reaches its maximum level, but at 12 months greatest remyelination , the MI tends to recover.

had a MI between 0.70 and 0.80, with a mean of 0.72"

0.07 being considered as a standard parameter which statis-tically differed from the MI in experimental groups. Onemonth after the SCC, the MI was higher but the highest MI

Ž .was observed at 2 months after the injury 0.90"0.03 .Afterwards, the MI was progressively smaller according to

Ž .the remyelination process Table 3 .Ž .There was a statistical difference p-0.02 between

Ž . ŽMI at 2 greatest demyelination and 12 greatest remyeli-.nation months after the SCC. Two months after the injury,

more than 60% of the axons was nonmyelinated and 12months after the contusion, only 30% of the axons had no

Ž .myelin sheath Fig. 5 .

4. Discussion

The reasons for the remyelinative relative failure in theCNS are poorly understood and this makes it difficult todevelop experimental strategies to promote efficient re-myelination in the SC. In the present study, the resultsobtained show the time course for plastic and remyelina-tive SC capabilities. These could be useful for understand-ing the moment at which different therapeutic strategiescan be used after a severe SCC injury without any interfer-ence or blockage of the spontaneous plasticity process ofthe system.

4.1. Plasticity

It has previously been described that lesioned axons intransected mammal SC, exhibit regrowth but just in an

w xabortive way 50 . This may be due to oligodendrocytesand central myelin as inhibitory substrates for axonal

w xgrowth 51–53 . They strongly contrast with the Schwanncells and the peripheral myelin that do not have theseinhibitory characteristics. During the present study, periph-eral Schwann cells and not oligodendrocytes were respon-sible for most of the remyelinative process. This explain,at least in part, that plasticity at the present experimentalmodel was manifested by the formation of a large number

Ž .of small new nerve fibers 0.5 mm or less in diameter ,considered as axon collaterals or sprouts with a high

Ž .correlation coefficient Spearman’s Rho tests0.83 . Thismeans that there were more axon collaterals as time elapsedafter the SCC. Recently, other authors have shown thataxon injury in the SC produces sprouts into the damaged

w xarea 35,36 . Nevertheless, physiological implications ofthis phenomenon should be investigated with the aim toknow if these small nerve processes have functional prof-itable effects.

On the other hand, in previous studies with contusionlesion models, the myelinated fiber loss has been described

w xas extensive and ranging between 87 and 99% 11 . In thisstudy, severe lesions were specifically made and the esti-mated myelinated fibers loss was 95.1%. In other words,an average of only 4.9% of the total population survivedthe lesion. The increase by 1.4% in the axon populationseen at 12 months may be due to some axon collateralshaving increased their diameter when they were myeli-nated and thus, were classified as axons rather than to anincrease per se of the axon number population.

4.2. Myelin

Myelin loss can result in a significant clinical deficitw x61 . This was originally thought to be permanent, similar


to axonal destruction. Currently, it is well know that thew xCNS retains its capacity to remyelinate axons 19,39,44

and some workers in the field sustain that even in severeSC injury, some recovery is the rule rather than the

w xexception 16,17,61 which could involve myelin regenera-w xtion 60 . However, the process requires a long period ofŽ .time at least a year in the present study and it may be due

to either the presence of inhibitory elements preventingmyelin repair or the absence of cells or factors necessary

w xfor new myelin synthesis 14,39,44,45,48 .In the peripheral nervous system, remyelination by

Schwann cells takes place rapidly and efficiently, stronglycontrasting with the inability of the CNS oligodendrocytes

w xto remyelinate under most circumstances 11,34 . AlthoughSchwann cells are able to remyelinate central axons, it iswell known that their migration is restricted by astrocytesw x12 , and eventually astrocytes seem to be responsible for

w xtheir expulsion from the CNS parenchyma 2,7,20 . How-ever, during the current study the most frequently myelina-tion pattern observed was characteristic of that producedby Schwann cells. Furthermore, remyelination by Schwanncells took place throughout all the experimental time courseand even at 12 months after the injury. This may be due tothe fact that the lesion was so severe that the breakage ofthe blood brain barrier cannot be totally repaired by theastrocytes, which leads to a continue Schwann cells migra-tion to the SC.

The Schwann cell myelination pattern was identified bythe presence of a thin cytoplasmic rim around the entireperimeter of the ensheathed axon, the presence of thenucleus and cytoplasm in the sheath surrounding the axon

w xand a basal lamina around the Schwann cells 46 . Further-more, Schwann cell myelinated fibers were usually sur-rounded by a clear extracellular space contrasting witholigodendrocytes myelinated fibers usually embedded in a

w xmore or less dense astrocytic field 11 .

4.3. Myelination index

The MI has been measured in different nerve structuresof several species and it appears that the myelinated fibershave an appropriate conduction when the MI is between0.60 and 0.80 with specific variations between differentspecies and between different nerve structures in the same

w xspecies 27,58 . In the present study, the MI obtained fromthe control group was 0.72"0.07. Two months after thelesion when the greatest demyelination was observed, theMI was far from the standard parameters. However, at 12months when remyelination by Schwann cells was impor-tant, the MI obtained was in close proximity to the MInecessary for appropriate nerve conduction.

On the other hand, it has been shown that the thicknessof the myelin sheath in animals without lesions increaseswith the enlargement of the axon diameter, maintaining a

wrelatively constant MI for fibers of all dimensions 22–

x24,29 . However, we found that some small-diameter ax-ons had myelin sheaths as thick as those seen in somelarge axons. This could be explained by the fact thatSchwann cells produce myelin sheaths thicker than oligo-dendrocytes do and in the present study most of the axonswere myelinated by Schwann cells. Furthermore, severallarge diameter axons had thin sheaths of myelin character-istic of smaller fibers which may be explained in termsthat the remyelination at this specific moment could havebeen partial. In the same way, the decrease in the propor-tion of small axons 6 months after the SCC could beexplained because when an axon is myelinated, its diame-

w xter increases 59 .According to our results, we do not feel the MI recov-

ery trend is due to an increase in the total number of axonsor in their corresponding MI. As previously discussed,1.4% of the axon population growth seen at 12 monthsmay be due to some myelinated axon collaterals increasedtheir diameter and thus being classified as axons ratherthan an increase per se in the number of axons. Neitherdoes it seem likely that this recovery has directly dependedon the increased myelin sheath thickness without increas-ing the number of myelinated axons since in axon popula-tions, very similar in number, the percentage of demyeli-nated fibers 2 months after the SCC was 64% and at 12months it was 30%. Hence, and because the rate ofSchwann cell myelinated axons was higher by far than therate of oligodendrocyte myelinated axons, and in somecases, only Schwann cells were seen to be the cause of thisprocess, we feel the increased number of Schwann cellmyelinated axons was responsible for the MI recoverytrend.

The results obtained in this study differ from those ofother authors that assert that remyelination in the CNS isabortive and limited. The differences can be explained onthe basis that some studies had rather subjective demyeli-

w xnation and remyelination evaluations 4,38 , while othersw xhad short experimental course times 17,25 , were carried

w xout in different species models 10,37 or evaluated differ-w xent pathologic processes 15,18,47,56 . Nonetheless, these

are CNS demyelinating pathologies too.In fact, functional recovery after a CNS lesion requires

w xfor the myelin to regenerate 60 , so with the aim to designrationale therapeutic strategies, we have to clearly under-stand the spontaneous regenerative mechanisms of the SCand not negatively interfere with them.

Currently, there are many experimental therapeutic pro-posals for the therapeutic SC injury bandling. Nonetheless,there is a need to develop strategies designed to hastenmyelin repair. One could consist in diminishing the initialdamage to the myelin sheaths with pharmacological pro-tecting treatments and the other, could be in increasing theinherent remyelinative capability of the system using en-riched transplants with the intention of repairing the dam-aged myelin sheaths, but considering the spontaneous plas-tic and regenerative processes of the SC.


Acknowledgements

The authors wish to thank Armando Zepeda, ElenaOliva, Adrian Rondan, Silvia Antuna and Gerardo Torresfor their help with the electron microscope, Manuel Perez´for his help with computational programs and Tomas Cruz´and Mario Garcıa for their photographic work. Hermelinda´Salgado-Ceballos holds a scholarship from The National

Ž .Council of Science and Technology CONACYT , contractnumber 80959.

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