Retrograde Activation of the Extrinsic Apoptotic Pathway in Spinal …downloads.hindawi.com/journals/bmri/2017/5953674.pdf · 2019-07-30 · BioMedResearchInternational 5 I1 I3 I4

Research ArticleRetrograde Activation of the Extrinsic ApoptoticPathway in Spinal-Projecting Neurons after a CompleteSpinal Cord Injury in Lampreys

Antoacuten Barreiro-Iglesias1 Daniel Sobrido-Cameaacuten1 andMichael I Shifman2

1Department of Functional Biology CIBUS Faculty of Biology Universidade de Santiago de Compostela15782 Santiago de Compostela Spain2Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation) Temple University School of Medicine3500 North Broad Street Philadelphia PA 19140 USA

Correspondence should be addressed to Michael I Shifman mshifmantempleedu

Received 31 July 2017 Accepted 25 October 2017 Published 19 November 2017

Academic Editor Antoni Camins

Copyright copy 2017 Anton Barreiro-Iglesias et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Spinal cord injury (SCI) is a devastating condition that leads to permanent disability because injured axons do not regenerateacross the trauma zone to reconnect to their targets A prerequisite for axonal regeneration will be the prevention of retrogradedegeneration that could lead to neuronal death However the specific molecular mechanisms of axotomy-induced degenerationof spinal-projecting neurons have not been elucidated yet In lampreys SCI induces the apoptotic death of identifiable descendingneurons that are ldquobad regeneratorspoor survivorsrdquo after SCI Here we investigated the apoptotic process activated in identifiabledescending neurons of lampreys after SCI For this we studied caspase activation by using fluorochrome-labeled inhibitors ofcaspases the degeneration of spinal-projecting neurons using Fluro-Jade C staining and the involvement of the intrinsic apoptoticpathway by means of cytochrome c and V120572 double immunofluorescence Our results provide evidence that after SCI bad-regenerating spinal cord-projecting neurons slowly degenerate and that the extrinsic pathway of apoptosis is involved in this processExperiments using the microtubule stabilizer Taxol showed that caspase-8 signaling is retrogradely transported by microtubulesfrom the site of axotomy to the neuronal soma Preventing the activation of this process could be an important therapeutic approachafter SCI in mammals

1 Introduction

In humans as in the other mammals spinal cord injury(SCI) causes permanent disability In mammals one of themain reasons for the failure of recovery is caused by theinability of axotomized axons to regenerate across the injurysite to reconnect to their targets An important goal of currentresearch aiming to develop a therapy for SCI is to promotethe regeneration of damaged axons [1] An important pre-requisite for axonal regeneration would be the prevention ofretrograde degeneration that could cause neuronal death oratrophy impeding the activation of axonal regrowth Severaltypes of central nervous system (CNS) neurons die after theysuffer axonal damage For example retinal ganglion cells [2ndash4] and motoneurons [5 6] die after axotomy However there

is still some controversy whether spinal cord (SC) projectingneurons of the brain ofmammals die after SCI Several studieshave shown the death of at least some brain neurons afterSCI (opossum [7] rats [8ndash12] humans [13]) However intwo recent studies using rats no evidence was found for thedeath of corticospinal neurons after SCI [14] and suggestedthat these neurons suffer atrophy after SCI but do not die[15] The death or degeneration (atrophy) of descendingneurons following SCI seems to involve an apoptotic processThis is suggested by the appearance of TUNEL staining andactivated caspase-3 immunoreactivity in descending neuronsof the brain (pontine reticular neurons [10] corticospinalneurons [9 11]) But more work is needed to fully understandthe molecular mechanisms that control the degeneration ofdescending neurons of the brain following a traumatic SCI

HindawiBioMed Research InternationalVolume 2017 Article ID 5953674 12 pageshttpsdoiorg10115520175953674

2 BioMed Research International

In contrast to mammals lampreys recover normalappearing locomotion spontaneously several weeks after acomplete SCI [16]This occurs due to the occurrence of plasticchanges [17ndash19] and regenerative processes [20ndash22] in spinalcircuits after the injury Descending neurons of lampreys areable to regenerate their axons after a complete SC transection[23 24] and a large percentage of spinal axons regener-ate in their correct paths [16 25] Moreover regenerateddescending axons are able to form new functional synapsiswith neurons below the site of injury [25ndash27] Among thereticulospinal neurons of the lamprey brain there are 36identifiable giant descending neurons [23] whose axonsproject almost the entire length of the SC These include theMauthner neurons which have crossed descending axonsand several pairs of Muller cells which have ipsilateraldescending axons Interestingly these identifiable descendingneurons vary greatly in their regenerative abilities followingSCI [23] Some of these neurons are considered ldquogood regen-eratorsrdquo (ie they regenerate their axonmore than 55 of thetimes) and others are considered ldquobad regeneratorsrdquo (ie theyregenerate their axon less than 30 of the times) Thuslampreys offer a model where there is an opportunity tostudy both enhancement and inhibition of regeneration inthe same preparation An additional advantage of the sealamprey model of SCI is that the identifiable descendingneurons and their descending axons can be visualized in vivoand in CNS whole-mounts due to the transparency of thelamprey brain This allows to study the intrinsic molecularprocesses that determine the fate of axotomized neuronsfollowing SCI For example recent studies have shown thatthe differential expression of axonal guidance receptors ingood or bad regenerators can explain at least in part theirdifferent regenerative abilities [22]

We and others have recently reported that a complete SCIinduces the delayed death of lamprey descending neuronsthat at earlier times after injury had been identified asbad regenerators [28 29] The occurrence of cell death wasdetermined based on the disappearance of Nissl stainingthe loss of neurofilament expression the absence of labelingwhen using retrograde tracers [28] and the early stainingof these neurons with Fluoro-Jade C (FJC) [29] which is amarker for degenerating neurons In addition the appearanceof TUNEL staining [28] and activated caspases [30ndash32] inthe soma of axotomized descending neurons suggested thattheir death after SCI is apoptotic A recent report by Barreiro-Iglesias and Shifman (2015) has shown that caspase activationin the cell perikarya of bad-regenerating descending neuronsof lampreys is preceded by the initial activation of caspasesin the axotomized axons at the lesion site within the firsthours after the injury This suggests that the degenerativeprocess is initiated at spinal levels These data indicatethat the identifiable descending neurons known to be ldquobadregeneratorsrdquo (ie the M1 M2 M3 I1 I2 Mth B1 B3and B4 descending neurons) suffer a process of slow anddelayed degeneration after SCI and therefore are also ldquopoorsurvivorsrdquoThus lampreys are a convenient vertebrate modelfor the in vivo study of molecular mechanisms underlying thedeath or degeneration of descending neurons after SCI

Apoptosis is a process that occurs normally during devel-opment and aging and as a homeostatic mechanism to main-tain cell populations in tissues but that can be also activatedafter cell damage [33] In apoptosis caspases are responsiblefor proteolytic cleavages that lead to cell disassembly (effectorcaspases) or that are involved in regulatory events (initiatorcaspases) [34] Research indicates that there are two mainapoptotic pathways the extrinsic or death receptor pathwayand the intrinsic or mitochondrial pathway The intrinsicpathway is activated by genomic and metabolic stress thepresence of unfolded proteins and other factors that leadto permeabilization of the outer membrane of mitochondriaand the release of apoptotic proteins mainly cytochrome cinto the cytosol Apoptosis initiation through the intrinsicpathway usually leads to the formation of the apoptosomecomplex and the activation of the initiator caspase-9 [34]Theextrinsic or death receptor pathway involves the activation ofdeath receptors This process leads to activation of initiatorcaspase-8 or caspase-10 [34] Recently we adapted the useof fluorochrome-labeled inhibitors of caspases (FLICA) todetect the activation of caspases in whole-mount prepa-rations of the sea lamprey brain following SCI [30] Weobserved that activated caspase-8 labeling appears in thesoma of ldquopoor survivorrdquo descending neurons within 2 weeksfollowing a SCI [30 31] and that the appearance of activatedcaspase-8 in the cell body was preceded by its activationin the injured axon rostral to the site of injury [31] Thissuggests that the extrinsic pathway of apoptosis is activatedin descending neurons after axotomy In the present studywe aimed to (1) further define the activation of the extrinsicapoptotic pathway in identifiable reticulospinal neurons afterSCI (2) study the possible activation of the intrinsic apoptoticpathway and (3) determine how the injury signals get to thecell body Our results provide additional evidence that afterSCI the bad-regenerating spinal-projecting neurons slowlydegenerate that only the extrinsic and not the intrinsic path-way of apoptosis is involved in this process and that caspase-8signaling is transported to the soma of descending neurons bymicrotubules

2 Material and Methods

21 Animals Wild type larval sea lampreys (PetromyzonmarinusL) 10ndash14 cm in length (4ndash7 years old) were obtainedfrom streams feeding Lake Michigan (USA) or from theRiverUlla (Spain) andmaintained in aerated freshwater tanksat 16∘C until the day of use Experiments were approvedby the Institutional Animal Care and Use Committee atTemple University and the by the Bioethics Committee atthe University of Santiago de Compostela and the Consellerıado Medio Rural e do Mar of the Xunta de Galicia (codeJLPVIId Spain) and were performed in accordance withEuropean Union and Spanish guidelines on animal care andexperimentation

22 Complete SC Transection Before experiments animals(119899 = 57) were deeply anesthetized by immersion in lampreyRinger solution containing 01 tricaine methanesulfonate(ScienceLab Houston TX USA) Complete SC transections

BioMed Research International 3

were performed as previously described [35] Briefly the SCwas exposed from the dorsal midline at the level of the fifthgill Complete transection of the SCwas performedwith Cas-troviejo scissors and the transectionwas confirmed under thestereomicroscope After surgery animals were kept on ice for1 hour allowing the wound to air dry Each transected animalwas examined 24 hours after surgery to confirm that therewas no movement caudal to the lesion site A transectionwas considered complete if the animal could move only itshead and body rostral to the lesion Animals were allowedto recover in aerated fresh water tanks at room temperaturefor different times (2 weeks 4 weeks 6 weeks 10 weeksor 4 months)

23 FJC Staining in Whole-Mounted Brain PreparationsFluoro-Jade C is a polyanionic fluorescein derivative andis commonly used in neuroscience to stain degeneratingneurons in the central nervous system regardless of specificinsult or mechanism of cell death [36 37] In the presentstudy FJC staining was used to further confirm that spinal-projecting neurons of lampreys slowly degenerate after spinalcord transection as shown previously with other markers ofcell death and degeneration [28]

Brains of control animals (119899 = 3) without a complete SCtransection (unlesioned) and brains of animals 4months (119899 =5) after the SC transection were removed in ice-cold Ringerand the dorsal choroid plexus covering the 4th ventricle wasremovedThe posterior and cerebrotectal commissures of thebrain were cut along the dorsal midline and the alar plateswere deflected laterally and pinned flat to a small strip ofSylgard (Dow Corning Co USA) Brains were fixed in 4paraformaldehyde (PFA) in phosphate buffered saline (PBS)for 2 hours at room temperature and washed on a nutatorfor 2 hours in PBS containing 2 Tween 20 Then brainswere immersed in 80 ethanol solution containing 1NaOHfor 5 minutes then transferred to 70 ethanol solution for2 minutes and rinsed in water for 2 minutes The brainswere incubated in 006 potassium permanganate solutionfor 10min and rinsed in water for 2 minutes The properdilution was made by making a 001 stock solution of FJCdye (Chemicon Temecula CA USA) in distilled water andthen adding 1mL of the stock solution to 99mL of 01 aceticacidThe brains were immersed in the staining solution for 25minutes and rinsed in water 3 times for 1 minute each Afterwashes brains were mounted on Superfrost Plus glass slides(Fisher Scientific MA USA) and coverslipped using Prolong(Invitrogen USA) as an antifade reagent Control brains wereprocessed in parallel with brains of animals 4 months afterinjury

24 Detection of Activated Caspases in Whole-MountedBrain Preparations The Image-iT LIVEGreen Poly CaspasesDetection Kit (Cat number I35104 Invitrogen USA) Image-iT LIVE Green Caspase-3 and -7 Detection Kit (Cat numberI35106 Invitrogen) and the Image-iT LIVE Green Caspase-8Detection Kit (Cat number I35105 Invitrogen) were used todetect activated caspases in identified reticulospinal neuronsof larval sea lampreys after a complete SC transection Thesekits contain 1 vial (component A of the kit) of the lyophilized

FLICA reagent (FAM-VAD-FMK for the detection of allactivated caspases FAM-DEVD-FMK for the detection ofactivated caspase-3 and caspase-7 and FAM-LETD-FMK forthe specific detection of activated caspase-8) Experimentswere done as previously described [30 31] Experiments forthe detection of all activated caspases were performed inanimals 2 weeks after the complete SC transection (119899 = 5)Experiments for the detection of activated caspase-8 were inanimals 4 weeks (119899 = 4) after the complete SC transectionExperiments for detection of activated caspase-3 and caspase-7 were done in control unlesioned animals (119899 = 5) and in ani-mals 6 weeks (119899 = 3) and 10 weeks (119899 = 4) after the completeSC transection

25 Double Cytochrome c and Complex V120572 Immunofluo-rescence in Whole-Mounted Brain Preparations Brains ofcontrol unlesioned animals (119899 = 4) and animals 2 weeks(119899 = 5) and 4 weeks (119899 = 5) after the complete SC transectionwere dissected fixed and processed as for the FJC staining(see above) After PFA fixation brains were washed on anutator at room temperature twice (1 hour each wash) in PBScontaining 2 Tween 20 and then twice (30 minutes eachwash) inmaleate buffer containing 02Tween 20Thebrainswere immersed for 1 hour in maleate buffer containing 15normal goat serum (MitoSciences Eugene Oregon USA)and incubated in the same solution containing a mouseanti-cytochrome c monoclonal IgG2a antibody (1 500 clone37BA11 catalog number MSA07 MitoSciences) and a mouseanti-complex V120572 (ATP synthase subunit alpha) monoclonalIgG2b antibody (1 500 clone 15H4C4 Cat number MSA07MitoSciences) for 2 days at 4∘C After washes as above thebrains were incubated in the same solution containing a goatanti-mouse IgG2a antibody conjugated to fluorescein isothio-cyanate (FITC) (1 500 Cat number MSA07 MitoSciences)and a goat anti-mouse IgG2b antibody conjugated to TexasRed (1 500 Cat number MSA07 MitoSciences) overnightat 4∘C Then the brains were rinsed in PBS containing01 Tween 20 mounted on Superfrost Plus glass slides(Fisher Scientific MA USA) and coverslipped using Prolong(Invitrogen) as an antifade reagent

The specificity of the anti-cytochrome c antibody wastested by Western blot using larval sea lamprey total proteinfrom combined spinal cord and brain samples homogenates(119899 = 2) For extract preparation the sample was lysedin TNN buffer (50mM Tris 150mM NaCl and 05 NP-40) with protease inhibitor cocktail (Sigma) The proteinconcentration was measured by the Bradford assay 30mglysate was loaded per well for SDS-PAGE and proteinswere transferred onto PVDF or nitrocellulose membranesWestern analysis was conducted by incubation with the anti-cytochrome c antibody (1 1000) overnight at 4∘C followed byincubation with HRP-tagged secondary anti-mouse antibody(1 5000 1 h at room temperature Americana Qualex SanClemente CA USA) Proteins were visualized using ECL(GE Health Sciences Piscataway NJ USA) The antibodyrecognized a single band of about 13 kDa (Figure 5(d))which corresponds to the molecular size of cytochromec of mammalian species Colocalization of cytochrome cand complex V120572 immunoreactivities in neurons of normal

4 BioMed Research International

animals (see Results) further confirmed the specificity of bothantibodies and the validity of the monoclonal anti-complexV120572 antibody as a mitochondrial marker As a control forsecondary antibodies the incubation with primary antibod-ies was omitted No immunoreactivity was observed in theseexperiments

26 Taxol (Placlitaxel) Treatment Immediately after thecomplete SC transection a small piece of Gelfoam soakedwith 10 120583L of either 1mM Taxol (Molecular Probes EugeneOR USA) in ethanol (119899 = 7) or 10 120583L of ethanol alone(119899 = 5) was placed on the top of the SC at the site of injuryAnimals recovered for 1 or 2 weeks and their brainsspinalcords were processed for detection of activated caspase-8 asabove Control animals that did not receive Taxol treatmentwere always processed in parallel with experimental animalstreated with Taxol

27 Imaging and Preparation of Figures Photomicrographswere taken using a Nikon Eclipse 80i microscope equippedwith a CoolSNAP ES (Roper Scientific USA) camera orwith a spectral confocal microscope (model TCS-SP2 LeicaWetzlar Germany) Images were always taken under thesame microscope conditions for control or treated animalsQuantification of mean fluorescent intensity of each identi-fiable neuron was done using the histogram function of theFiji software For the preparation of figures brightness andcontrast were minimally adjusted using Adobe PhotoshopCC software and lettering was added

28 Statistical Analyses Statistical analysis was carried outusing Prism 6 (GraphPad software La Jolla CA) Datawere presented as mean plusmn SEM Normality of the data wasdetermined by the DrsquoAgostino-Pearson normality test Thecorrelation (Pearson test) between fluorescence intensity ofthe FLICA labeling (present results) and the regenerativeability of the descending neurons [23] was analyzed Differ-ences in fluorescence intensity between control and Taxoltreated animalswere analyzed bymeans of a two-tailed pairedStudentrsquos 119905-test

3 Results

31 Levels of Activated Caspases 2 Weeks after a Complete SCTransection Correlate Significantly with the Long-Term Regen-erative Ability of Identifiable Neurons In previous studies[23 38] some reticulospinal neurons of the sea lamprey wereidentified as ldquobad regeneratorsrdquo (M1 M2 M3 I1 I2 Mth B1B2 B3 and B4) because their axons had a low probability ofregenerating after axotomy due to a complete SC transectionIn later studies almost the same cells were identified as ldquopoorsurvivorrdquo neurons because they have a high likelihood ofdegenerating and dying after axotomy (the M1 M2 M3 I1I2 Mth B1 B3 and B4 neurons) [28]

Also previous studies revealed that bad regeneratorpoorsurvivor neurons show high levels of activated caspases in thefirst 2 weeks following a complete SC transection as revealedby FLICA labeling [30ndash32] and that intense FLICA labelingand TUNEL staining are observed in bad regenerators several

weeks after a complete SC transection [32] However thesestudies never established a statistical correlation between theintensity of FLICA labeling in the first 2 weeks after the injuryand the long-term regenerative ability of the individuallyidentifiable neurons Here we used a poly-caspase FLICAreagent (FAM-VAD-FMK) to detect all activated caspases2 weeks following a complete SC transection (Figures 1(a)and 1(b)) and correlated the levels of activated caspases inidentifiable neurons with their known regenerative ability (of times inwhich a specific neuron regenerates its axon acrossthe lesion site based on the results of [23]) This revealed asignificant correlation (119901 = 00044 Pearson test) betweenthe level of activated caspases (fluorescence intensity) andthe known regenerative ability of the identifiable neurons(Figure 1(c))

32 Long-Term Detection of Specific Activated Caspases inBad Regenerators In previous studies we reported intenseFLICA labeling 2 weeks after lesion in bad regenerators usingthe FLICA reagent specific for the detection of activatedcaspase-8 (FAM-LETD-FMK) [30] Here we extended ouranalyses and observed that at one month posttransectionFAM-LETD-FMK labeling is still observed in ldquopoor survivorrdquoSC projecting neurons of the brainstem (M1 M2 M3 I1 I2Mth B1 B3 and B4 Figure 2)

Incubation of the brains with the FLICA reagent FAM-DEVD-FMK revealed the presence of activated caspase-3or caspase-7 in identified SC projecting brainstem neuronsat 10 weeks posttransection (Figure 3) but not in neuronsof untransected control animals (Figure 3(a)) Little or nolabeling was observed 6 weeks posttransection (not shown)Intense FAM-DEVD-FMK labeling was observed in the cellbodies of identified spinal-projecting neurons known to beldquopoor survivorsrdquo (the M1 M2 M3 I1 I2 Mth B1 B3and B4 neurons) Interestingly the morphology of theseneurons differed from that of the same neurons in controlanimals The neurons seemed to have suffered atrophy andhad fewer dendrites than normal neurons (Figure 3(b)ndash3(d))The delay between the detection of activated caspase-8 andthe detection of activated caspase-3caspase-7 may accountfor the slow process of cell death observed in these neuronsafter axotomy [28]

33 Staining with Fluoro-Jade C Is Consistent with ProtractedDegeneration of Axotomized Spinal-Projecting Neurons FJCstaining (Figure 4) was not observed in identifiable spinal-projecting neurons of control unlesioned animals (Fig-ure 4(b)) Four months after the SC transection intense FJClabeling was observed in swollen identifiable reticulospinalneurons previously identified as ldquobad regeneratorsrdquo (M1 M2M3 I1 I2 Mth B1 B3 and B4) (Figure 4(a)) This wasconsistent with the results of Busch and Morgan [29] andextended the period in which FJC staining was detectedto 4 months confirming that spinal-projecting neurons oflampreys degenerate very slowly after SCI

34 Cytochrome c Is Not Released fromMitochondria of Spinal-Projecting Neurons after the Complete SC Transection Thepresence of activated caspase-8 in descending neurons 2 [30]

BioMed Research International 5

I1

I3

I4

(a)

Mth

MtＢ

(b)

rege

nera

tion

R square = 04271Y = minus07135 lowast X + 1077

100

80

60

40

20

0

0 50 100 150 200

I3I5

I4 B6

B5

I6M1 B2

I2

B1M2 M3

MthB4

B3 I1

Fluorescence intensity

MtＢ

(c)

Figure 1 Confocal photomicrographs of dorsal views of whole-mounted brains of larval sea lampreys showing FAM-VAD-FMK labeling inaxotomized identified spinal-projecting neurons 2weeks after SCI (a) I1 I3 and I4 neurons (b)Mth andMth1015840 neuronsNote that fluorescenceintensity in I1 andMth neurons is higher than I3 I4 andMth1015840 neurons Rostral is to the top and the ventricle is to right in all figures (c) Linearregression analysis shows the inverse correlation between the regenerative ability of identified SC projecting neurons and their fluorescenceintensity reflecting caspase activation (95 confidence intervals for slopeminus1168 tominus02588 Syx = 2050119901 = 00044)The dotted lines indicatethe 95 confidence interval Identity of RS neurons is shown adjacent to their corresponding data points I1 I3 and I4 Muller cells of theisthmic region Mth Mauthner cell Mth1015840 auxiliary Mauthner cell Scale bars 50120583m

and 4 (see above) weeks after a complete SC transectionsuggests that the extrinsic apoptotic pathway is activated indescending neurons after axotomy To determine whetherthe intrinsic pathway is also involved in the death of theseneurons after axotomy we performed double immunofluo-rescence experiments in whole-mounted brain preparationsusing anti-cytochrome c and anti-complex V120572 antibodiesCytochrome c release from mitochondria is a key step inthe development of the intrinsic apoptotic pathway becauseit allows the formation of the apoptosome complex andsubsequent activation of caspase-9 (see above)

In immunofluorescence experiments colocalization ofcytochrome c and complex V120572 immunoreactivities wasobserved as a cluster of punctuate staining in the cellbodies of all identifiable spinal-projecting neurons in controlunlesioned animals (Figure 5(a)) and in animals 2 and 4weeks posttransection (Figures 5(b) and 5(c)) This showsthat at time points after SCI in which activated caspase-8 is

already detected in the cell bodies of ldquopoor survivorrdquo neurons([30] present results) cytochrome c has not been releasedfrom the mitochondria

35 Taxol Treatment Prevents Caspase-8 Retrograde Acti-vation in Spinal-Projecting Neurons after SCI The initialactivation of caspase-8 at the site of injury followed by itsprogressively proximal appearance toward the cell bodies ofldquopoor survivorrdquo SC projecting neurons suggests that eitherthe signals that activate caspase-8 andor activated caspase-8 itself is retrogradely transported from the site of axotomyto the cell bodies [31] To determine whether the centripetalmovement of caspase-8 activation depends on microtubule-based retrograde transport from the site of axotomy weapplied themicrotubule stabilizer Taxol (placlitaxel) to the SCat the site of a complete transection Pieces of Gelfoam soakedeither with ethanol (control) or with a solution of 1mMTaxolin ethanol were inserted into the lesion site Brains from

6 BioMed Research International

M3

M2

4 weeks SCI

M1

(a)

I1

4 weeks SCI

(b)

Figure 2 Photomicrographs of dorsal views of whole-mounted brains of larval sea lampreys showing FAM-LETD-FMK labeling inaxotomized descending neurons 4 weeks after SCI ((a) and (b)) Rostral is to the top and the ventricle is to left in all figures I1 Mullercell of the isthmic region M1ndashM3 Muller cells 1 to 4 Scale bars 50 120583m in (a) and 20 120583m in (b)

Mth

B3

B1

B4

No SCI

(a)

Mth

B3

B4

10 weeks SCI

(b)

I1I5

10 weeks SCI

(c)

M3

10 weeks SCI

(d)

Figure 3 Photomicrographs of dorsal views of the whole-mounted brain of larval sea lampreys showing the presence of intense FAM-DEVD-FMK labeling in ldquopoor survivorrdquo spinal-projecting neurons 10weeks after a complete spinal cord transection ((b)ndash(d)) as compared to controlswithout injury (a) Note the absence of labeling in an I5 cell in (c) Rostral is to the top and the ventricle to the left in all figures B1 B3 andB4 Muller cells of the bulbar region I1 and I5 Muller cells of the isthmic region M3 Muller cell 3 Mth Mauthner cell Scale bars 50 120583m in(a)ndash(c) and 15 120583m in (d)

BioMed Research International 7

Mth Mth

4 months SCI

B3B3

B1

⋆

(a)

Mth

No SCI

Mth

B3 B4 B3

B1B1 ⋆

(b)

Figure 4 Photomicrographs of dorsal views of whole-mounted brains of larval sea lampreys showing Fluoro-Jade C staining in swollenreticulospinal neurons 4 months after a complete SC transection (a) Note the absence of labeling in control animals without SC transection(b) The star indicates the location of the ventricle Rostral is to the top in the figures B1 B3 and B4 Muller cells of the bulbar region MthMauthner cell Scale bars 125120583m

Mth

No SCI

Cc + V

(a)

Mth

2 weeks SCI

Cc + V

(b)

4 weeks SCI

Mth

B3

B4

Cc + V

(c)

38

25

18

13

10

(kD

a)

Cytochrome c

(d)

Figure 5 Photomicrographs of dorsal views of whole-mounted brains of larval sea lampreys showing colocalization of cytochrome c andcomplex V120572 immunoreactivities (arrows) in identifiable reticulospinal neurons of control animals (a) and animals 2 (b) and 4 (c) weeks aftera complete SCI Rostral is to the top and the ventricle to the left in all figures (d) Western blot showing that the anti-cytochrome c antibodyrecognizes a single band of the expected molecular weight for cytochrome c (about 13 kDa) B3 and B4 Muller cells of the bulbar region Cccytochrome c Mth Mauthner cell Scale bars 20120583m in (a) and (b) and 15 120583m in (c)

these animals were analyzed 2 weeks later for the presenceof activated caspase-8 as above (Figure 6) Intense FAM-LETD-FMK labeling was observed in ldquopoor survivorrdquo spinal-projecting neurons of control animals (Figures 6(b) and6(d)) but in animals treated with Taxol these neurons wereeither unlabeled or exhibited only veryweak labeling (Figures6(a) and 6(b)) Statistical analyses revealed a significantdifference in caspase-8 activation (fluorescence intensity)between control and Taxol treated animals (119901 lt 00001 two-tailed paired Studentrsquos 119905-test Figure 6(e))

To determine whether the Taxol treatment caused inhibi-tion of caspase-8 activation itself or inhibited microtubule-based retrograde transport of caspase-8 activity from thesite of axotomy some animals were studied 1 week post-transection (Figure 7) As in the animals studied 2 weeksafter injury no labeling was observed in spinal-projectingneurons (Figure 7(b)) However labeling was observed in thedescending axons close to the site of injury (Figure 7(a))This indicates that the Taxol treatment inhibited the transportof the signals that activate caspase-8 or the transport of

8 BioMed Research International

Mth

Taxol

B3 B1

B5

B4

(a)

Mth

B3B1

B4EtOH

(b)

Taxol

I1I2

(c)

I1

EtOH

(d)

150

100

50

0

Fluo

resc

ence

inte

nsity

M1 M2 M3 I1 I2 Mth B1 B3 B4Identifiable neurons

ControlTaxol

(e)

Figure 6 Photomicrographs of dorsal views of whole-mounted brains of larval sea lampreys showing reduced FAM-LETD-FMK labeling inidentifiable ldquopoor survivorrdquo SC projecting neurons after Taxol ((a) and (c)) treatment as compared with control animals treated with ethanolalone ((b) and (d)) (e) Graph revealing a significant difference (119901 lt 00001 two-tailed paired Studentrsquos 119905-test) in fluorescence intensity of theFAM-LETD-FMK labeling in identifiable neurons of control and Taxol treated animals Rostral is to the top in all figures The ventricle is tothe right in all figures B1 and B3ndashB5 Muller cells of the bulbar region I1 and I2 Muller cells of the isthmic region Mth Mauthner cell Scalebars 50 120583m in (a) (c) and (d) and 25 120583m in (b)

activated caspase-8 itself but did not prevent the initialprocess of caspase-8 activation

4 Discussion

Present findings together with previous results [28ndash32] showthat in lampreys axotomy due to SCI consistently causesretrograde degeneration and apoptotic death in individuallyidentified spinal cord-projecting neuron that is they areldquopoor survivorsrdquo and that these are the same cells that areldquobad regeneratorsrdquo [23] The persistence of FJC staining 4months after the injury together with the late detection ofactivated caspase-3caspase-7 (present results) and TUNELstaining [28] in these neurons prior to their disappearance

indicates that axotomy in the SC causes the activation of avery slow process of cell death in SC projecting neurons Thepresence of activated caspase-8 and the lack of cytochromec release indicate that the extrinsic apoptotic pathway is themain mediator of this cell death process after axotomy Ourresults also suggest that the centripetal activation of caspasesdepends on microtubule-based retrograde transport of stresssignals

Studies in mammals have shown that a proportion ofSC projecting neurons die or undergo atrophy after damageto their axon at the spinal level and that this may dependon activation of an apoptotic process (see Introduction)Clearly prevention of this process would be a prerequisite fordesigning therapies to promote regeneration of descending

BioMed Research International 9

Taxol

(a)

Mth

B1

B3B4

Taxol

(b)

Figure 7 Photomicrographs of dorsal views of the whole-mounted spinal cord (a) and brain (b) of a larval sea lamprey treated with Taxolshowing the presence of FAM-LETD-FMK labeling in the tip of descending axons 1 week after treatment (arrows in (a)) and the absence oflabeling in identifiable descending neurons of the brain (b) Rostral is to the right in (a) and to the top in (b)The ventricle is to the left in (b)B1 B3 and B4 Muller cells of the bulbar region Mth Mauthner cell Scale bars 40120583m in (a) and 50120583m in (b)

pathways after SCI [39] Therefore it is of great importanceto understand the molecular processes that lead to theretrograde degeneration of injured descending pathways afterSCI This could also inform research in other types of CNSinjuries like optic nerve injuries or stroke where apoptoticprocesses are also activated [40ndash45] Previous results inlampreys showed that axotomy at the spinal level initiallyleads to activation of caspase-8 in the injured axon at the siteof injury and subsequently in the cell body of descendingneurons [30] This is in agreement with reports showingthat axotomy induces caspase-8 activation in retinal ganglion[44 45] and olfactory receptor [46] neurons Other studieshave also revealed the importance of caspase-2 and caspase-6 in causing the death of retinal ganglion cells followingoptic nerve transections [41ndash43] Experiments using themicrotubule stabilizer Taxol show that both in SC projectingneurons (present results) and in olfactory neurons [46] theretrograde activation of caspase-8 in the perikaryon afteraxotomydepends onmicrotubule-based retrograde transportfrom the injured axon In olfactory neurons the appearanceof activated caspase-8 in the cell body depended on themicrotubule-based retrograde transport of activated caspase-8 probably by its association with dynactin p150Glued [46]We cannot rule out the possibility that other retrogradesignals may promote activation of local procaspase-8 in thecell body and along the axon of SC projecting neurons Asin olfactory neurons [46] axotomy of SC projecting neuronsin lampreys caused sequential activation of caspase-8 andcaspase-3 or caspase-7 (present results) Our results indicatethat Taxol or other microtubule stabilizers like epothiloneB may be a beneficial therapeutic option after SCI not onlyto promote axonal regeneration as previously reported foraxons in the mammalian SC [47 48] and optic nerve [49]but also to avoid the retrograde degeneration of SC projectingneurons by preventing the retrograde transport of caspase-8 or other stress signals Of note the retrograde activationof neuronal degeneration might be slow in lampreys due toslower microtubule dynamics that occur in cold-living fishes[50 51]

Studies in mammals also reported increased levels ofactivated caspase-8 in the SC after injury [52ndash55] Becausethese analyses used immunoblots of SC homogenates [52ndash55] it is not known if the activation of caspase-8 occurredin intraspinal cells in descending axons or in both Neutral-ization of the proapoptotic cytokines tumor necrosis factor-related apoptosis-inducing ligand [55] or tumor necrosisfactor-alpha [55] promoted a decrease in caspase-8 activationafter SCI Dependence receptors could also be importantplayers in the initiation of apoptotic processes after CNSdamage Dependence receptors like Neogenin or UNC5receptors trigger extrinsic apoptotic processes in the absenceof their ligands [56 57] Recent work has shown that an inhi-bition of Neogenin localization in lipid rafts promotes sur-vival and axonal growth after SC or optic nerve injuries [58]Interestingly recent work in lampreys has also shown thatknock down of Neogenin in descending neurons of lampreyspromotes axonal regeneration [22] Another study in lam-preys has also shown that knockdown of the small GTPaseRhoA enhances axon regeneration and inhibits retrogradeapoptosis following SCI [59] The results of our experimentsin lampreys could also be translated to mammalian injureddescending axons because mechanisms of caspase-activatedapoptosis are evolutionary conserved [60 61] The sea lam-prey could be an interesting in vivo animal model to studythe role of proapoptotic cytokines dependence receptors orother molecules like RhoA in the intra-axonal activation ofcaspase-8 after SCI

Abbreviations Used in the Figures

B1ndashB6 Muller cells of the bulbar region (middlerhombencephalic reticular nucleus)

Cc Cytochrome cEtOh EthanolI1ndashI6 Muller cells of the isthmic regionM1ndashM4 Muller cells 1 to 4Mth Mauthner cellMth1015840 Auxiliary Mauthner cellSCI Spinal cord injury

10 BioMed Research International

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

The authors sincerely acknowledge Professor ME Selzer forhis critical reading of the manuscriptThey also acknowledgethe Servicio de Microscopıa and thank Dr Mercedes RivasCascallar (Universidade de Santiago de Compostela) forconfocal microscope facilities and assistance This work wassupported by grants from the Spanish Ministry of Economyand Competitiveness and the European Regional Develop-ment Fund 2007ndash2013 (BFU2014-56300-P) and the Xuntade Galicia (GPC2014030) Anton Barreiro-Iglesias was sup-ported by a grant from the Xunta de Galicia (2016-PG008)and by Shriners Hospitals for Children Postdoctoral Fellow-ship Michael I Shifman was funded by Shriners ResearchFoundation Grant (SHC-85310)

References

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[2] M Berkelaar D B Clarke Y-C Wang G M Bray and A JAguayo ldquoAxotomy results in delayed death and apoptosis ofretinal ganglion cells in adult ratsrdquoThe Journal of Neurosciencevol 14 no 7 pp 4368ndash4374 1994

[3] P Chaudhary F Ahmed P Quebada and S C Sharma ldquoCas-pase inhibitors block the retinal ganglion cell death followingoptic nerve transectionrdquo Brain Research vol 67 no 1 pp 36ndash45 1999

[4] P Kermer N Klocker M Labes S Thomsen A Srinivasanand M Bahr ldquoActivation of caspase-3 in axotomized rat retinalganglion cells in vivordquo FEBS Letters vol 453 no 3 pp 361ndash3641999

[5] J L Vanderluit L TMcPhail K J L Fernandes et al ldquoCaspase-3 is activated following axotomy of neonatal facial motoneuronsand caspase-3 gene deletion delays axotomy-induced cell deathin rodentsrdquo European Journal of Neuroscience vol 12 no 10 pp3469ndash3480 2000

[6] Y-M Chan L-W Yick H K Yip K-F So R W OppenheimandWWu ldquoInhibition of caspases promotes long-term survivaland reinnervation by axotomized spinal motoneurons of dener-vatedmuscle in newborn ratsrdquo Experimental Neurology vol 181no 2 pp 190ndash203 2003

[7] E J Fry H B Stolp M A Lane K M Dziegielewska and N RSaunders ldquoRegeneration of Supraspinal Axons after CompleteTransection of theThoracic Spinal Cord in Neonatal Opossums(Monodelphis domestica)rdquo Journal of Comparative Neurologyvol 466 no 3 pp 422ndash444 2003

[8] E R Feringa and H L Vahlsing ldquoLabeled corticospinalneurons one year after spinal cord transectionrdquo NeuroscienceLetters vol 58 no 3 pp 283ndash286 1985

[9] B C Hains J A Black and S G Waxman ldquoPrimary corticalmotor neurons undergo apoptosis after axotomizing spinal cordinjuryrdquo Journal of Comparative Neurology vol 462 no 3 pp328ndash341 2003

[10] K L H Wu S H H Chan Y-M Chao and J Y H ChanldquoExpression of pro-inflammatory cytokine and caspase genes

promotes neuronal apoptosis in pontine reticular formationafter spinal cord transectionrdquo Neurobiology of Disease vol 14no 1 pp 19ndash31 2003

[11] B H Lee K H Lee U J Kim et al ldquoInjury in the spinal cordmay produce cell death in the brainrdquo Brain Research vol 1020no 1-2 pp 37ndash44 2004

[12] N Klapka S Hermanns G Straten et al ldquoSuppression offibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons rescue ofprimary motoneurons in somatosensory cortex and significantfunctional recoveryrdquo European Journal of Neuroscience vol 22no 12 pp 3047ndash3058 2005

[13] G Holmes and W P May ldquoOn the Exact Origin of thePyramidal Tracts in Man and other Mammalsrdquo Journal of theRoyal Society of Medicine vol 2 pp 92ndash100 1909

[14] J L Nielson I Sears-Kraxberger M K Strong J K Wong RWillenberg andO Steward ldquoUnexpected survival of neurons oforigin of the pyramidal tract after spinal cord injuryrdquoThe Jour-nal of Neuroscience vol 30 no 34 pp 11516ndash11528 2010

[15] J L Nielson M K Strong and O Steward ldquoA reassessmentof whether cortical motor neurons die following spinal cordinjuryrdquo Journal of Comparative Neurology vol 519 no 14 pp2852ndash2869 2011

[16] M C Rodicio and A Barreiro-Iglesias ldquoLampreys as an animalmodel in regeneration studies after spinal cord injuryrdquo Revistade Neurologıa vol 55 no 3 pp 157ndash166 2012

[17] M I Becker and D Parker ldquoChanges in functional propertiesand 5-HT modulation above and below a spinal transection inlampreyrdquo Frontiers in Neural Circuits vol 8 pp 1ndash18 2015

[18] M E Cornide-Petronio B Fernandez-Lopez A Barreiro-Iglesias and M C Rodicio ldquoTraumatic injury induces changesin the expression of the serotonin 1A receptor in the spinal cordof lampreysrdquo Neuropharmacology vol 77 pp 369ndash378 2014

[19] B Fernandez-Lopez A Barreiro-Iglesias and M C RodicioldquoAnatomical recovery of the spinal glutamatergic system follow-ing a complete spinal cord injury in lampreysrdquo Scientific Reportsvol 6 Article ID 37786 2016

[20] SM Fogerson A J van Brummen D J Busch et al ldquoReducingsynuclein accumulation improves neuronal survival after spinalcord injuryrdquo Experimental Neurology vol 278 pp 105ndash115 2016

[21] J Chen C Laramore and M I Shifman ldquoDifferential expres-sion of HDACs and KATs in high and low regeneration capacityneurons during spinal cord regenerationrdquo Experimental Neurol-ogy vol 280 pp 50ndash59 2016

[22] J Chen C Laramore and M I Shifman ldquoThe expressionof chemorepulsive guidance receptors and the regenerativeabilities of spinal-projecting neurons after spinal cord injuryrdquoNeuroscience vol 341 pp 95ndash111 2017

[23] A J Jacobs G P Swain J A Snedeker D S Pijak L JGladstone andM E Selzer ldquoRecovery of neurofilament expres-sion selectively in regenerating reticulospinal neuronsrdquo TheJournal of Neuroscience vol 17 no 13 pp 5206ndash5220 1997

[24] M E Cornide-Petronio M S Ruiz A Barreiro-Iglesias andM C Rodicio ldquoSpontaneous regeneration of the serotonergicdescending innervation in the sea lamprey after spinal cordinjuryrdquo Journal of Neurotrauma vol 28 no 12 pp 2535ndash25402011

[25] M E Selzer ldquoMechanisms of functional recovery and regener-ation after spinal cord transection in larval sea lampreyrdquo TheJournal of Physiology vol 277 no 1 pp 395ndash408 1978

BioMed Research International 11

[26] C M Rovainen ldquoRegeneration of Muller and Mauthner axonsafter spinal transection in larval lampreysrdquo Journal of Compar-ative Neurology vol 168 no 4 pp 545ndash554 1976

[27] M R Wood and M J Cohen ldquoSynaptic regeneration inidentified neurons of the lamprey spinal cordrdquo Science vol 206no 4416 pp 344ndash347 1979

[28] M I Shifman G Zhang and M E Selzer ldquoDelayed deathof identified reticulospinal neurons after spinal cord injury inlampreysrdquo Journal of Comparative Neurology vol 510 no 3 pp269ndash282 2008

[29] D J Busch and J R Morgan ldquoSynuclein accumulation isassociated with cell-specific neuronal death after spinal cordinjuryrdquo Journal of Comparative Neurology vol 520 no 8 pp1751ndash1771 2012

[30] A Barreiro-Iglesias and M I Shifman ldquoUse of fluorochrome-labeled inhibitors of caspases to detect neuronal apoptosis in thewhole-mounted lamprey brain after spinal cord injuryrdquo EnzymeResearch vol 2012 Article ID 835731 7 pages 2012

[31] A Barreiro-Iglesias and M I Shifman ldquoDetection of ActivatedCaspase-8 In Injured Spinal Axons By Using Fluorochrome-Labeled Inhibitors Of Caspases (FLICA)rdquoMethods inMolecularBiology vol 1254 pp 329ndash339 2015

[32] J Hu G Zhang and M E Selzer ldquoActivated caspase detectionin living tissue combined with subsequent retrograde labelingimmunohistochemistry or in situ hybridization in whole-mounted lamprey brainsrdquo Journal of Neuroscience Methods vol220 no 1 pp 92ndash98 2013

[33] S Elmore ldquoApoptosis a review of programmed cell deathrdquoToxicologic Pathology vol 35 no 4 pp 495ndash516 2007

[34] M S Ola M Nawaz and H Ahsan ldquoRole of Bcl-2 familyproteins and caspases in the regulation of apoptosisrdquoMolecularand Cellular Biochemistry vol 351 no 1-2 pp 41ndash58 2011

[35] A Barreiro-Iglesias G Zhang M E Selzer M I Shifman andM E Selzer ldquoComplete spinal cord injury and brain dissectionprotocol for subsequent wholemount in situ hybridization inlarval sea lampreyrdquo Journal of Visualized Experiments no 92Article ID e51494 2014

[36] L C Schmued C Albertson andW Slikker Jr ldquoFluoro-Jade Anovel fluorochrome for the sensitive and reliable histochemicallocalization of neuronal degenerationrdquo Brain Research vol 751no 1 pp 37ndash46 1997

[37] L C Schmued C C Stowers A C Scallet and L Xu ldquoFluoro-Jade C results in ultra high resolution and contrast labeling ofdegenerating neuronsrdquo Brain Research vol 1035 no 1 pp 24ndash31 2005

[38] G R Davis and A D McClellan ldquoExtent and time courseof restoration of descending brainstem projections in spinalcord-transected lampreyrdquo Journal of Comparative Neurologyvol 344 no 1 pp 65ndash82 1994

[39] A Barreiro-Iglesias ldquoBad regenerators die after spinal cordinjury Insights from lampreysrdquo Neural Regeneration Researchvol 10 no 1 pp 25ndash27 2015

[40] X Chen X Zhang L Xue C HaoW Liao andQWan ldquoTreat-ment with enriched environment reduces neuronal apoptosisin the periinfarct cortex after cerebral ischemiareperfusioninjuryrdquo Cellular Physiology and Biochemistry vol 41 no 4 pp1445ndash1456 2017

[41] Z AhmedH KalinskiM Berry et al ldquoOcular neuroprotectionby siRNA targeting caspase-2rdquo Cell Death amp Disease vol 2 no6 article e173 2011

[42] V Vigneswara M Berry A Logan and Z Ahmed ldquoPharmaco-logical inhibition of caspase-2 protects axotomised retinal gan-glion cells from apoptosis in adult ratsrdquo PLoS ONE vol 7 no 12Article ID e53473 2012

[43] VVigneswaraNAkpanM Berry A Logan CM Troy andZAhmed ldquoCombined suppression of CASP2 andCASP6 protectsretinal ganglion cells from apoptosis and promotes axon regen-eration through CNTF-mediated JAKSTAT signallingrdquo Brainvol 137 no 6 pp 1656ndash1675 2014

[44] J H Weishaupt R Diem P Kermer S Krajewski J CReed and M Bahr ldquoContribution of caspase-8 to apoptosis ofaxotomized rat retinal ganglion cells in vivordquo Neurobiology ofDisease vol 13 no 2 pp 124ndash135 2003

[45] P P Monnier P M DrsquoOnofrio M Magharious et al ldquoInvolve-ment of caspase-6 and caspase-8 in neuronal apoptosis and theregenerative failure of injured retinal ganglion cellsrdquoThe Journalof Neuroscience vol 31 no 29 pp 10494ndash10505 2011

[46] C Carson M Saleh F W Fung D W Nicholson and AJ Roskams ldquoAxonal dynactin p150Glued transports caspase-8to drive retrograde olfactory receptor neuron apoptosisrdquo TheJournal of Neuroscience vol 25 no 26 pp 6092ndash6104 2005

[47] F Hellal AHurtado J Ruschel et al ldquoMicrotubule stabilizationreduces scarring and causes axon regeneration after spinal cordinjuryrdquo Science vol 331 no 6019 pp 928ndash931 2011

[48] J Ruschel F Hellal K C Flynn et al ldquoSystemic administrationof epothilone B promotes axon regeneration after spinal cordinjuryrdquo Science vol 348 no 6232 pp 347ndash352 2015

[49] V Sengottuvel M Leibinger M Pfreimer A Andreadaki andD Fischer ldquoTaxol facilitates axon regeneration in the matureCNSrdquoThe Journal of Neuroscience vol 31 no 7 pp 2688ndash26992011

[50] M Billger M Wallin R C Williams and H W DetrichldquoDynamic instability of microtubules from cold-living fishesrdquoCell Motility and the Cytoskeleton vol 28 no 4 pp 327ndash3321994

[51] H W Detrich III ldquoMicrotubule assembly in cold-adaptedorganisms Functional properties and structural adaptations oftubulins from Antarctic fishesrdquo Comparative Biochemistry andPhysiology Part A Physiology vol 118 no 3 pp 501ndash513 1997

[52] S CashaW R Yu andMG Fehlings ldquoOligodendroglial apop-tosis occurs along degenerating axons and is associated withFAS and p75 expression following spinal cord injury in the ratrdquoNeuroscience vol 103 no 1 pp 203ndash218 2001

[53] S Casha W R Yu andM G Fehlings ldquoFAS deficiency reducesapoptosis spares axons and improves function after spinal cordinjuryrdquo Experimental Neurology vol 196 no 2 pp 390ndash4002005

[54] G Cantarella G Di Benedetto M Scollo et al ldquoNeutralizationof tumor necrosis factor-related apoptosis-inducing ligandreduces spinal cord injury damage in micerdquo Neuropsychophar-macology vol 35 no 6 pp 1302ndash1314 2010

[55] K-B Chen K Uchida H Nakajima et al ldquoTumor necrosisfactor-120572 antagonist reduces apoptosis of neurons and oligoden-droglia in rat spinal cord injuryrdquoThe Spine Journal vol 36 no17 pp 1350ndash1358 2011

[56] E Matsunaga and A Chedotal ldquoRepulsive guidancemoleculeneogenin A novel ligand-receptor system playingmultiple roles in neural developmentrdquo Development Growth ampDifferentiation vol 46 no 6 pp 481ndash486 2004

[57] X Tang S-W Jang M Okada et al ldquoNetrin-1 mediates neu-ronal survival through PIKE-L interactionwith the dependence

12 BioMed Research International

receptor UNC5Brdquo Nature Cell Biology vol 10 no 6 pp 698ndash706 2008

[58] N G Tassew A J Mothe A P Shabanzadeh et al ldquoModifyinglipid rafts promotes regeneration and functional recoveryrdquo CellReports vol 8 no 4 pp 1146ndash1159 2014

[59] J Hu G Zhang W Rodemer L-Q Jin M Shifman and M ESelzer ldquoThe role of RhoA in retrograde neuronal death and axonregeneration after spinal cord injuryrdquo Neurobiology of Diseasevol 98 pp 25ndash35 2017

[60] K Sakamaki and Y Satou ldquoCaspases Evolutionary aspects oftheir functions in vertebratesrdquo Journal of Fish Biology vol 74no 4 pp 727ndash753 2009

[61] M Boyce A Degterev and J Yuan ldquoCaspases An ancientcellular sword of Damoclesrdquo Cell Death amp Differentiation vol11 no 1 pp 29ndash37 2004

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