thoracolumbar penetrating spinal cord injury ( sci ) often produces motor and sensory alterations in hindlimbs . promising pharmacological treatments and treadmill locomotion training
are used for inducing restoration of locomotion and spinal reflexes after contusive , compressive lesions or by a penetrating sci [ 24 ] .
locomotion disturbances occur in concordance with the type of injury and the spinal cord area suffering the trauma [ 5 , 6 ] .
axonal and neuronal death is an important secondary effect after a penetrating injury in brain and spinal cord lesions [ 7 , 8 ] .
tamoxifen has been shown to be an effective treatment to brain and spinal cord injuries ; it has been proposed as an inflammatory response modulator and participates in locomotion recovery after a sci [ 2 , 8 , 9 ] .
tamoxifen is a selective estrogen receptor modulator ( serm ) acting on - and -estrogen receptors ; also , it is been shown to prevent demyelination and promote differentiation to oligodendrocytes from multipotential cells in the cerebral cortex .
it is unknown whether tamoxifen exerts neuroprotective effects in cats after a sci . in this study , experiments were made on cats with a t13-l1 level spinal cord hemisection and the effects of tamoxifen were assessed by evaluating the survival of neurons in the spinal cord , axonal myelin preservation , or remyelination and by analyzing the kinematic angular displacement of both hindlimbs during unrestricted gait .
adult male cats were used in this study ( laboratory animal center of the guadalajara university , 3.54  kg , n = 9 ) .
all the procedures described here were performed with the guidelines contained in the national institutes of health guide for the care and use of laboratory animals ( usa ) and with the ethical considerations stipulated in the experimental animal treatment on the official mexican norm ( nom-062-zoo-1999 ) .
experiments were approved by the ethics committee for research and biosafety ( universidad de guadalajara ) .
cats were divided in three groups : intact ( int , n = 3 ) , injured and treated with tamoxifen ( iwt , n = 3 ) , and injured without tamoxifen ( iwot , n = 3 ) .
a prophylactic antibiotic treatment was given ( gentamicin 2  mg / kg i.m . ) .
the cats were anesthetized with ketamine ( 10  mg / kg i.p . ) and xylazine ( 1  mg / kg i.p . ) for performing spinal cord injury .
the dorsal surface of the t12 vertebra was exposed and the apophysis was removed with a surgical gouge .
a microdrill was used for opening the right lamina , and a surgical blade ( hergom number 11 ) for hemisecting the spinal cord segment .
after surgery , a drop of medical grade cyanoacrylate was applied on the dura mater and bone wax was used to cover the vertebra .
animals were treated for three days with postoperative antibiotics ( gentamicin 2  mg / kg i.m . ) .
iwt cats received tamoxifen ( 1  mg / kg i.p . ) at days 0 , 1 , and 2 dai ( days after injury ) .
animals had free access to water and food and were housed one per cage ( 1  m height  1  m wide  1  m tall ) to allow them to move freely .
prior to the surgery , animals were trained to walk through a transparent acrylic passway ( 200  cm long  50  cm high  20  cm width ) daily during one week , in order to record a basal walk kinematic in cats .
video contrasting dots were placed in the iliac crest , hip ( i.e. , greater trochanter ) , knee , ankle ( i.e. , lateral malleolus ) , and fifth metatarsal phalangeal joints . the marks were placed in both hindlimbs and videotaped with a 30-frame - per - second video camera ( sony fdr - ax100 ) .  total video converter 
( shareware ) was used for decomposing the video into individual frames and the cartesian coordinates of each joint mark were determined by the image j software ( scion corporation , nih ) .
subsequently , the joint mark coordinates were introduced in a labview environment computer program ( developed in cinvestav , ipn , mxico ) .
line graphs were constructed to illustrate the hindlimb movements from at least 3 consecutive strides .
the computer program also calculates the hip and knee joint angles in a movement sequence executed by the ipsilateral and contralateral hindlimbs during strides .
the joint angular displacement ( jad ) was calculated from the difference between the maximum and minimal angular values of each stride .
in addition , hindlimb plm was analyzed by determining the angle between a line drawn from the iliac crest to the ankle and the y - axis ( figure 1 ) .
control kinematics data was obtained prior to spinal cord injury in all subjects ; subsequently , the experimental values were acquired at 4 , 16 , and 30 dai in iwt and iwot groups .
all data were normalized and were graphically represented as a percent of the angular displacement . at 30 dai cats were deeply anesthetized ( pentobarbital euthanasia dose 50  mg / kg i.m . ) and intracardially perfused with 500  ml of 0.1  m phosphate buffered saline ( pbs ) containing 0.5  ml of heparin ( 1000  u.i./ml ) , followed by 500  ml of a fixative solution ( 4% paraformaldehyde in 0.1  m pbs ) .
the spinal cord segment t13-l1 was extracted and placed in the fixative solution during 24 hours and then washed in 0.1  m pbs for another 24 hours .
the spinal cord segment was cryoprotected in 0.1  m pbs containing 30% of sucrose and 30% ethylene glycol for 72 hours .
subsequently , the spinal cord segment was divided in three regions : ( 1 ) injury site ( is ) , ( 2 ) cuts initiating 200  m rostral from the injury site ( rfi ) , and ( 3 ) cuts initiating caudally from the injury site ( cfi ) .
the tissue was embedded in leica tissue freezing medium and representative 15  m thick coronal cuts of each section were obtained from the t13-l1 segment using a leica cm1850 cryostat .
nine coronal cuts were placed per each slide ; two slides per section were stained and analyzed . a hematoxylin and eosin ( h&e ) staining protocol was used for evaluating the histopathology status ( observing whether wallerian degeneration occurred ; and the lesion similarity ) of the spinal cord in int , iwt , and iwot cats .
hematoxylin solution was used for 5 minutes per slide and alcoholic eosin y solution ( 0.5% eosin in 90% ethanol ) for 30 seconds ; slides were washed with tap water for 5 minutes after staining .
toluidine blue staining protocol was used for assessing myelin thickness after sci in all groups .
o - toluidine hydrochloride solution ( 3% o - toluidine in 0.1  m acetate - acetic buffer ph 5.0 ) was used for 5 minutes per slice ; 2-minute washing was performed with tap water .
subsequently , an ethanol dehydration protocol was applied and the slices were mounted with entellan. the ventral white matter was visualized with light microscopy ( olympus , bx51w1 ) and photographed with a high resolution camera ( canon eos rebel t3 ) .
a 300  m square microscopic field was delimitated for establishing a qualitative white matter axon observation using the portable olympus image pro plus software v 6.0 .
the myelin area was determined in axons from the ventral white matter ( cat 1 , n = 30 ; cat 2 , n = 20 ; cat 3 , n = 30 , per analyzed section ) ; the inclusion criterions were maximum diameter of 15  m and a round - like morphology .
this study was performed using an image software analyzer ( motic images plus 2.0 ) .
the axonal myelin area was determined by establishing a perimeter trace around the outer myelin sheet and a second perimeter trace around the inside myelin sheet .
the nonspecific antigen binding sites were blocked by a 10% normal goat serum for 1 hour at room temperature .
the sections were incubated for 18 hours with the primary antibody at 4c : anti - fox3 for neurons , previously known as neun   ( abcam 104225 , 1  :  1000 ) .
the slices were washed between incubations ( three times ) in pbs for 10  min .
after primary antibody , the slices were incubated with the goat polyclonal secondary antibody : anti - igg alexa fluor 488 conjugated anti - rabbit ( invitrogen a-11008 , 1  :  1000 ) during 2 hours at room temperature .
after the secondary antibody , tissues were incubated with 4,6-diamidino-2-phenylindole ( dapi ) ( molecular probes d3571 , 1  :  100 ) for 5 minutes .
nine coronal cuts per slide were placed manually , and three slides were evaluated in each level ( cat 1 : 18 cuts for rfi , 18 cuts for is , and 18 cuts for cfi ; cat 2 : 18 cuts for rfi , 18 cuts for is , and 18 cuts for cfi ; cat 3 : 18 cuts for rfi , 18 cuts for is , and 18 cuts for cfi ) ; the ethanol dehydration protocol was applied and sealed with entellan .
neurons were counted using a digital image software ( portable olympus image pro plus software v 6.0 ) , adapted to a fluorescence microscope .
six microscopic fields ( 500  m ) were studied in the dorsal horn ( dh ; rexed lamina i and ii ) , ventral horn ( vh ; rexed lamina viii and iv ) , and periaqueductal zone ( paz ; rexed lamina x ) , ipsilateral and contralateral to the injury .
all data is expressed as mean  sd . neurons ( n = 18 cuts per slide ) and myelin morphometric ( n = 30 ) data was analyzed using a nonparametric kruskal - wallis test and mann - whitney u test for multiple comparisons .
kinematic assessment was analyzed using a nonparametric friedman test followed by wilcoxon post hoc test for multiple specific comparisons .
ibm spss ( release 20.0.0 ) software was used for statistical tests and graphs were made using statistical software ( graphpad prism 6.0 ) .
a schematic drawing of the spinal cord damage for treated and untreated cats is illustrated in figures 2(b ) and 2(c ) .
the spinal cord slices from intact cats show defined borders between gray and white matter .
the gray matter showed a purple homogeneous color definition ( basophilic staining , figure 2(d ) ) .
considerable damage , involving a large portion of the ipsilateral white and gray matter , is observed in the iwot spinal cord tissue .
the border between white and gray matter disappeared ; the contralateral white matter showed hypochromic staining and the gray matter has poor basophilic and eosinophilic staining features showing abnormal characteristics ( figure 2(e ) ) . in iwt cats ,
also tissue hallows as well as poor delimitation of white and gray matter were observed .
in contrast , the contralateral side has preserved normal characteristics ( figure 2(f ) ) . a qualitative assessment of the ventral white matter axons was made .
symmetrical axon morphology and a purple homogeneous color definition were observed in intact white matter , and there is no tissue inflammation or hollows ( figures 3(a ) , 3(f ) , and 3(k ) ) . at the is in the iwot cats , axons in the ventral white matter of the ipsilateral side appeared with spheroid morphology ( figure 3(g ) ) . in the contralateral side ,
a hypochromic staining with wallerian degeneration characteristics was observed ( figure 3(i ) ) . in iwt cats , axons appeared with a clearly defined morphology which was similar to int cats axons at the ipsi- and contralateral side ( figures 3(h ) and 3(j ) ) .
similar normal stained characteristics were observed in cfi ( figures 3(l)3(o ) ) . at rfi ,
myelin in int , iwt , and iwot ventral axons is illustrated in figures 4(a)4(o ) ; intact axon myelin thickness in ventral axons was considered as 100% .
ipsilateral is axonal myelin was reduced to 29.7  2.4% in iwot cats ; in iwt cats , myelin was reduced to 64  2.0% with a significant differences between groups ( p < 0.001 ) ( figure 4(q ) ) .
contralateral side myelin was reduced to 28.7  6.7% in iwot cats and to 65  11.06% in iwt cats ( p < 0.001 ) .
similar results were obtained in the rfi and cfi regions ( figures 4(p ) and 4(r ) , resp . ) .
iwt and iwot groups showed significant ipsilateral and contralateral statistical changes in the myelin percentages in comparison with int cats .
these changes occurred in is , rfi , and cfi regions ( figures 4(p)4(r ) ) . although the previously mentioned results were normalized to percentage values , real myelin thickness values
fox-3/dapi positive cells were counted for evaluating spinal cord neuronal survival in int , iwo , and iwt cats . at 30 dai ,
an increase of the neuronal survival in tamoxifen treated cats was observed , particularly in the ventral zone ( figures 5(a)5(r ) ) .
the number of neurons in int , iwt , and iwot groups in ipsilateral and contralateral side at the is , rfi , and cfi regions is plotted in figure 6 . at 30 dai ,
vh neurons decreased in is , rfi , and cfi regions on both sides in iwt and iwot cats .
the number of ipsilateral and contralateral neurons was partially recovered by tamoxifen treatment ( table 1 , figures 6(c ) , 6(f ) , and 6(i ) ) . spinal cord damage reduced paz neurons in all studied regions , in treated and untreated cats ( table 1 , figures 6(b ) , 6(e ) , and 6(h ) ) .
dh neurons quantities did not change in the is and the cfi site , in ipsilateral or contralateral spinal cord .
this partial neuronal preservation occurred in treated and untreated cats ( figures 6(d ) and 6(g ) ) .
it is important to mention that dh neurons were significantly reduced in the rfi region in untreated cats but recovered in treated cats in ipsilateral and contralateral spinal cord regions ( figure 6(a ) ) .
three consecutive ipsilateral hindlimb ( ihl ) and contralateral hindlimb ( chl ) strides were recorded ( figure 7 ) . in normal stepping cats ,
the ihl and chl executed symmetrical steps with well - defined stance and swing phases ( figures 7(a ) and 7(h ) ) .
in contrast , at 4 dai , ihl exhibited asymmetrical steps and limb dragging in both iwt and iwot cats ( figures 7(b ) and 7(c ) ) . at 16 dai
, we observed that iwt and iwot cats partially recovered their gait locomotor movements ( figures 7(d ) and 7(e ) ) . at 30 dai ,
treated and untreated cats ilh showed a normal stepping sequence , indicating complete gait locomotion recovery ( figures 7(f ) and 7(g ) ) . at 4 dai , iwot cats exhibited an altered stride and oscillatory - like hip movements ( as a compensatory process for maintaining gait locomotion and avoiding animal downfall ) ( figure 7(i ) ) .
stride alteration continued at 16 dai ( figure 7(k ) ) and partial locomotion recovery is observed at 30 dai ( figure 7(m ) ) . in iwt cats , a locomotion recovery was observed at day 16 and it was clearly maintained until 30 dai ( figures 7(l)7(n ) ) .
the hip angular displacements in the ihl and chl in iwt and iwot cats are illustrated in figure 8 .
ipsilateral hip ad values in both groups of animals ( iwt and iwot cats ) decreased approximately by 40% at 4 dai but returned to their base values at 16 and 30 dai ( figure 8(a ) and table 2 ) . whereas the iwot cat contralateral hip exhibited a significant decrement in jad values ( nearly 50% ) at 4 and 16 dai ,
in contrast , the contralateral hip of iwt cats showed a considerable increment in jad values ( nearly to 150% ; table 2 ) at 4 and 16 dai and recovered their base values at 30 dai ( tables 2 and 3 , figure 8(b ) ) . at 4 and 16
dai , the ipsilateral knee of iwot cats exhibited a statistically significant decrease in jad values , approximately 50% , whereas the knee joint of iwt cats showed an increment in jad values of approximately 50% .
both groups ( iwt and iwot cats ) returned to their base values at 30 dai ( figure 8(c ) ) .
no significant changes were observed in the contralateral knee ( tables 2 and 3 , figure 8(d ) ) . in hindlimb
plm , both ipsi- and contralateral had values recovered faster in cats treated with tamoxifen , as compared to nontreated animals . at 4 dai
, ihl angular displacement values showed a statistically significant decrease in both , iwt and iwot groups .
iwt cat 's pendulum - like movement values showed partial recovery at 16 dai and nearly a 100% recovery at 30 dai . at 30 dai ,
pendulum - like movement of iwot cats recovered by nearly 90% ( tables 2 and 3 , figure 8(e ) ) . at 4 and 16
dai , ihl angular displacement values in iwot cats increased by 50% and 20% , respectively . at 30 dai , angular displacement values
, no differences occurred in the contralateral plm ( tables 2 and 3 , figure 8(f ) ) .
the penetrating injury applied in our model produced a degeneration process characterized by spheroid axons and tissue hollows occurring in ipsilateral as well as in the contralateral side of the injury .
a wallerian degenerative process seems to be occurring on both sides in accordance with previous results , reporting an axonal wallerian degeneration after a spinal cord injury [ 12 , 13 ] .
tamoxifen is serm acting on the -estradiol estrogen receptors ( er ) [ 14 , 15 ] .
in addition , it produces neuronal protection after brain penetrating injury   and reduces microglia reactivity . in rats
, tamoxifen reduces the inflammatory process after a spinal cord injury   and the neuronal death .
further experiments studying tamoxifen 's effects on neural inflammation process , neurotrophic factors , and inflammatory interleukins would be required for comparing tamoxifen 's effects in cats to those observed in rats .
contusion impact in a cat 's spinal cord produces an altered axonal morphology . in our study , tamoxifen reduced spheroid axons in the ipsilateral spinal cord in iwt cats , recovering a quasi - normal morphology .
this preservation could be favored by an inhibition of the glutamate excitotoxicity after the injury and also by an attenuation of inflammatory mediated damage [ 8 , 19 ] .
previous works indicate that tamoxifen promotes ng2 multipotent progenitor cell differentiation changing into oligodendrocytes and could favor remyelination after rat brain penetrating injury [ 7 , 21 ] .
rat spinal cord contusion produces deleterious effects in neurons at 24 hours [ 22 , 23 ] .
after one month , they were attributed to an inflammatory process . in iwot cats , at 30 dai , after the spinal cord injury , the number of positive fox-3 cells was reduced on both ipsilateral and contralateral sides . in iwt cats ,
the number of neurons in vh and dh is similar to the number in int cats .
this result could be related to the morphological er distribution in the thoracolumbar spinal cord , as reported in other animal species [ 2426 ] .
this effect could be attributed to a minor amount of er of the paz neurons .
it has been demonstrated that estrogen receptors are poorly expressed in periaqueductal neurons and are present mostly in the ventral horn in the female cat 's lumbosacral spinal cord .
further experiments must be performed for evaluating the spinal cord er distribution in male cats .
therefore , important contralateral plastic changes could be contributing to a rapid locomotion recovery but not through the paz neurons pathway . in this study we analyzed overground locomotion . at 16 dai
spinal cord injury models had been previously described [ 2931 ] . in these models ,
the locomotion recovery was related to the severity of the sci . in the present experiments , all cats suffered a similar damage at the t13-l1 level ( figure 2 ) .
therefore , the locomotion recovery by tamoxifen treatment was consistent because the damage of the spinal cord extension was similar in all cats . in the present experiments
, the locomotion onset might depend more on the undamaged contralateral spinal cord than a step training plasticity process , given that the cats were not trained for walking .
the kinematic parameters were altered during the first 16 dai in both hindlimbs but were partially or fully restored after 30 dai .
it is of interest that , in iwt cats , recovery to nearly normal values already occurred at 16 days ; therefore tamoxifen shortened the recovery time .
to the best of our knowledge , hemisected cats treated with tamoxifen and walking overground were studied for the first time .
locomotion recovery results observed in this study highlight the importance of preserving contralateral descending pathways for locomotion initiation and kinematic recuperation .
it would be of interest to study the effects of tamoxifen in relation to avoiding obstacles during a cat 's normal walking task .
a previous study sheds light on this question , in which they assessed hemisected spinal cord cats during overground walking while avoiding obstacles in their way .
in addition to what was previously stated , a contusion model in rats has been used for studying tamoxifen anti - inflammatory effects , but the kinematics in injured rats under tamoxifen treatment remain to be established [ 5 , 34 ] . at present time , we consider that a study must be made in which the combined treatment with tamoxifen and treadmill training to improve the outcome of hemisected animals should be carried out .
the current study demonstrates that hemisected spinal cord produces important locomotion alterations in both hindlimbs .
tamoxifen may be useful in several animal species as therapeutic treatment in spinal cord injury .
 we performed experiments in cats with a spinal cord penetrating hemisection at t13-l1 level , with and without tamoxifen treatment . 
 the results showed that the numbers of the ipsilateral and contralateral ventral horn neurons were reduced to less than half in the nontreated animals compared with the treated ones . 
 also , axons myelin sheet was preserved to almost normal values in treated cats . on the contrary , in the untreated animals , 
 their myelin sheet was reduced to 28% at 30 days after injury ( dai ) , in both the ipsilateral and contralateral regions of the spinal cord . additionally , we made hindlimb kinematics experiments to study the effects of tamoxifen on cat locomotion after the injury : at 4 , 16 , and 30 dai . 
 we observed that the ipsilateral hindlimb angular displacement ( ad ) of the pendulum - like movements ( plm ) during gait locomotion was recovered to almost normal values in treated cats . 
 contralateral plm acquired similar values to those obtained in intact cats . at 4 dai , untreated animals showed a compensatory increment of plm occurring in the contralateral hindlimb , which was partially recovered at 30 dai . 
 our findings indicate that tamoxifen exerts a neuroprotective effect and preserves or produces myelinated axons , which could benefit the locomotion recovery in injured cats . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions