dendritic spines are small protrusions that serve as a postsynaptic site for the 90% of the excitatory synapses in the cns . different kinds of dendritic spines were described based on their shape and their actin content in adult rat brains .
mushroom dendritic spines have stalks with a clear head differentiation , stubby spines are thick and have no neck , and thin spines are characterized as long and without neck . mushroom dendritic spines have a rich actin cytoskeleton network [ 1 , 2 ] , which is highly regulated by proteins that either stabilize the actin monomer ( g - actin ) , such as thymosin or profiling , arp2/3 , rho - gtpase kinase contractin , or prevent polymerization and convert several polymers into small fragments of actin such as cofilin and gelsolin .
several functions have been suggested for dendritic spines as they have been implicated in the mechanism of synaptic plasticity , learning and memory [ 47 ] , and protein translocation
. functional decline of dendritic spines is a consequence of synaptic loss in neurodegenerative disease and brain insults .
furthermore , processes such as loss of dendritic spines , dendritic pruning , and loss of synaptic proteins precede neuronal death in many neurodegenerative disorders [ 1012 ] . moreover , activation and impaired function of the ubiquitin - proteasome pathway is thought to contribute to a number of neurodegenerative disorders .
therefore , spine pathologies may be involved in different brain insults including hypoxia ischemia [ 1417 ] .
perinatal asphyxia ( pa ) is a serious clinical complication with high mortality and morbidity .
following pa , approximately 45% of newborn die and 25% have permanent neurological deficits including cerebral palsy , mental retardation and developmental delay , learning disabilities , visual and hearing problems , and different issues in school readiness [ 1924 ] . in previous works , we have observed several alterations in striatum and hippocampal areas after pa , such as high level of ubiquitinization in dendritic spines , reactive gliosis , alterations in dendritic microtubular organization , and modification in cytoskeleton organization [ 17 , 25 , 26 ] .
given that numerous reports support the idea that dendritic spines are the main site damaged during brain ischemia [ 25 , 27 ] , we aimed to investigate whether dendritic spine changes are a spread feature induced by pa .
for this purpose , we studied dendritic spine modifications in the stratum radiatum of ca1 hippocampal area .
  all procedures involving animals were approved by the institutional animal care and use committee at the university of buenos aires ( school of medicine ) and conducted according to the principles of the guide for the care and use of laboratory animals ( nih publications no .
sprague - dawley female rats in the fifteenth day of pregnancy were placed in individual cages and maintained on a 12  :  12  h light / dark cycle in a controlled temperature ( 21  2c ) and humidity ( 65  5% ) environment . the animals had access to food ( purina chow ) and tap water ad libitum .
one group of animals ( n = 10 ) was used as surrogate mothers , another group ( n = 10 ) was assigned to pa procedures .
eosin - phalloidin and phalloidin - alexa were purchased from invitrogen ( carlsbad , ca ) .
secondary antibodies against mouse were obtained from jackson immunoresearch laboratories ( west grove , pa ) .
paraformaldehyde , em grade glutaraldehyde , sodium cacodylate , and durcopan acm resin were obtained from electron microscopy sciences ( fort washington , pa ) ; special welled tissue culture plates were obtained from mattek ( ashland , ma ) .
ten full - term pregnant rats on gestational day 22 were anesthetized , rapidly decapitated , and the uterus horns were isolated through an abdominal incision and placed in a water bath at 37c for 19  min ( subsevere pa : n = 10 full - term pregnant rats ) [ 25 , 26 , 2931 ] .
we have used 19  min as the maximum time of pa because more than 20 minutes result in a survival rate lower than 3% .
following asphyxia , the uterus horns were rapidly opened , the pups were removed , the amniotic fluid was cleaned , and the pups were stimulated to breathe by performing tactile stimulation with pieces of medical wipes for a few minutes until regular breathing was established .
the umbilical cords were ligated , and the animals were left to recover for 1 hour under a heating lamp .
when their physiological conditions improved , they were given to surrogate mothers who had delivered normally within the past 24 hours .
the different groups of pups were marked and mixed with the surrogate mothers ' normal litters ( control animals ( ctl ) that were left undisturbed ) .
briefly , an intracardiac perfusion was performed with normal rat ringer 's at 35c followed by fixative under deep anaesthesia ( containing 50  mg / kg ketamine , 1  mg / kg rhompun and 5  mg / kg acetopromazine in sterile saline ) . for light microscopy analysis
, rats were perfused with 4% formaldehyde ( freshly made from paraformaldehyde ) in 0.1  m phosphate buffer , ph 7.2 .
the brain was removed and fixed for 2 additional hours in the same solution at 4c .
then , sections were embedded in durcupan acm resin . after removing the brain from the skull ,
it was postfixed in the same fixative during 2  h. coronal or sagital sections ( 5080  m ) were made with a vibratome ( leica ) .
some of these sections were stained with cresyl violet according to the procedures described in capani et al . .
vibratome sections were washed with 50  mm glycine - pbs containing 0.5% cold water fish gelatin to block nonspecific binding . following 30  min of washing
, the sections were incubated on a shaker , in a solution of 0.05% of eosin phalloidin-0.5% cold - water fish gelatin/50  mm glycine - pbs for 2  h at 4c . for light microscopic studies ,
phalloidin conjugated to alexa was also used because of its superior fluorescent quantum yield compared to eosin . as a negative control ,
tissue sections stained with eosin - phalloidin were mounted on glass - welled tissue culture dishes ( mat tek corp ) pretreated with polyethylenimine .
the slices were fixed again for 25  min with 2% glutaraldehyde in 0.1  m cacodylate buffer , rinsed in buffer for several minutes , and placed in 50  mm glycine and potassium cyanide in cacodylate buffer for an additional 5  min to reduce nonspecific staining .
photooxidation was performed on the zeiss axiovert described above , equipped with a 75-w xenon arc light source .
the samples were immersed in a solution of 2.8  mm diaminobenzidine in 0.1  m sodium cacodylate at 4c bubbled with pure o2 , final ph 7.4 , and then irradiated under conventional epifluorescence using a xenon lamp .
after 68  min , a brownish reaction product began to appear in place of the fluorescence .
following photooxidation , tissue sections were rinsed in 0.1  m sodium cacodylate several times and incubated for 30  min with 1% osmium tetroxide in 0.1  m sodium cacodylate , ph 7.2 .
after several washes in double - distilled h2o , the sections were dehydrated in an ascending ethanol series , flat - embedded in durcopan acm resin , and polymerized for 24  h at 60c .
serial thin sections ( 80100  nm ) were cut with reichert ultracut e ultramicrotome using glass knives and examined using a jeol 100cx electron microscope at 80100  kev .
one set of thin sections was poststained with a combination of uranyl acetate and lead citrate . for e - pta staining ,
sections were dehydrated in an ascending series of ethanol to 100% and stained for 1  h with 1% pta stained prepared by dissolving 0.1  g of pta in 10  ml of 100% ethanol and adding four drops of 95% ethanol .
the volume fraction of immunoreactive material for phalloidin was estimated using the point - counting method of weibel   and a grid delimiting 5000  m in the hippocampus .
percentage of reactive area was estimated using image j program ( image j 1.41 , nih , usa ) . for electron microscopy analysis sampling ,
all of the synapses that have the characteristic of mushrooms dendritic spines ( head larger than the neck ) were used in this study since mushrooms dendritic spines are the unique f - actin positive spines .
random fields of neuropil containing at least one synapse were photographed at 10000x magnification and analyzed at a total magnification of 30000x .
ca1 hippocampal specimens were selected for quantitative analyses based on the quality of e - pta staining and the degree of ultrastructural preservation , as determined from conventionally stained material from the same animals .
samples were analyzed from controls ( n = 4 ) and 19  min pa animals ( n = 8) .
tissue sections were cut at thickness of 100  nm and examined and photographed at 80  kev at a magnification of 8300x with a zeiss 109 electron microscope . for each animal , five micrographs were obtained from hippocampus . as described above , each negative was digitized into a pc computer .
using nih image 1.6 , psds were first manually outlined , and then the maximal thickness , minimum thickness , length , and total area of each psd were determined .
all synapses in which the postsynaptic density , intracleft line , and presynaptic grid were clearly visible were chosen for analysis .
the selection criterion resulted in the analysis of between 30 and 50  psds per animal for each hippocampus . for analysis
all of the synapses that have the characteristic of mushrooms type dendritic spines ( head larger than the neck ) were used in this study since mushrooms spines are the unique f - actin positive spines .
random fields of neuropil containing at least one synapse were photographed at 10000x magnification and analyzed at a total magnification of 30000x .
biochemical fractionation was performed as described previously by saraceno et al . using the whole dorsal hippocampus   ( ctl , n = 6 ; pa n = 6 ) .
dounce homogenates ( h ) of the pellets in ice cold tevp buffer ( 10  mm tris - hcl , ph 7.4 , 5  mm naf , 1  mm na3vo4 , 1  mm edta , and 1  mm egta , 1.25  g / ml pepstatin a , 10  g / ml leupeptin , 2.5  g / ml aproptionin , 0.5  mm pmsf ) containing 320  mm sucrose were centrifuged at 1000  g to remove nuclei and large debris ( p1 ) .
the supernatant ( s1 ) was centrifuged at 10.000  g for 10  min to obtain a crude synaptosomal fraction ( p2 ) and subsequently was lysed hypoosmotically and centrifuged at 45.000  g for 90  min to obtain a pellet of the synaptosomal membrane fraction ( lp1 ) . after each centrifugation
, the resulting pellet was rinsed briefly with ice cold tevp buffer before subsequent fractioning to avoid possible crossover contamination .
western blot analysis was carried out using lp1 fractions separated on 10% sds - page .
samples containing 50  g of protein from each group were applied to each lane .
after electrophoresis , proteins were transferred to polyvinylidene difluoride ( pvdf ) membrane as described previously [ 3638 ] .
the membranes were incubated with a primary antibody anti--actin ( sigma , 1  :  1000 ) overnight at 4c . then , after appropriate washing procedures , they were incubated with horseradish peroxidase - conjugated anti - mouse secondary antibody for 2 hours at room temperature .
the films were scanned , and the optical density of protein bands was quantified using gel pro analyzer software 3.1.00.00 ( media cybernetics , usa ) .
we used glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) as load controls [ 17 , 3941 ] .
statistical analyses were performed using the graphpad prism 5.03 for windows statistical package ( graphpad software , inc , san diego , ca , usa ) .
the study of nuclear morphology by cresyl violet staining showed that pa animals present clear nuclear condensation 4 months after injury respect to ctl animals in the stratum radiatum ca1 hippocampal area sections ( figure 1 ) .
statistical analyses showed alterations in pyramidal neurons of hippocampal ca1 area , showing an abundance of pyknotic nuclei in asphyctic animals as compared to control animals ( table 1 ) .
then , we employed neuronal nuclei ( neun ) immunolabeling to determine the nature of the cells presenting condensed nuclei ( figure 1 ) .
statistical analyses showed no significant difference in the number of neun+ nuclei in the ca1 hippocampal area of asphyctic animals respect to controls .
when we analyzed the cellular distribution of neun labeling , it was determinate that asphyctic animals showed a significant increase in the number of abnormal neun+ nuclei and a significant decrease in the number of normal neun+ nuclei in the ca1 hippocampal area compared with ctl group ( table 1 ) . to determine the morphology of these cells
morphological analyses showed that most cells presenting condensed nuclei evidence dark cytoplasm with rare vacuoles and compaction , a hypertrophic nucleolus , a nucleus with a festoon shape , and a twisted nuclear envelope , corresponding to neurons in degeneration [ 25 , 32 , 42 , 43 ] ( figure 1 ) .
osmium - lead - citrated staining showed no obvious alterations in the stratum radiatum ca1 hippocampal area sections from 4-month - old ctl and pa rats ( figure 2 ) .
presynaptic terminals , presynaptic vesicles , and ultrastructural organization of psd were intact ( figure 2 ) . on the other hand ,
e - pta immunostaining showed clear alterations in synapses of rats subjected to pa ( figure 3 ) .
following pa , the thickness of psd increased as compared to controls ( figure 3 ) .
there was also a general increment in the amount of e - pta - stained material in psd of pa animals compared to controls .
t student analyses for the area and length of the psd and for the minimum and maximum thickness of the psd were significant ( p < 0.05 ) .
post hoc tests revealed that the means of psd area was significantly bigger as compared to the ctl group ( p < 0.01 ) ( table 2 ) .
these inconsistencies between the osmium and e - pta staining may be attributable to the fact that general heavy metal staining obscures the synaptic modifications occurring in post asphyctic tissue .
in addition , it is possible that e - pta stains different components in the psd than osmium - uranium - lead methods . it has been known that psds stained with e - pta are shorter and probably wider than those stained with the osmium - heavy metal method .
e - pta preferentially stains protein(s ) rich in basic amino acid residues , including lysine , arginine , and histidine , such as collagen or histones .
in contrast , conventional heavy metal staining stains a wide type of lipids and cytoskeletal and cytoplasmic elements .
since both markers stain different components , this might explain why e - pta staining is more effective to detect the psds alterations than heavy metal - stained sections .
consistent with other studies in different models of ischemia [ 27 , 33 , 43 ] and using this long - term pa model , we did not observe any alterations in the subcellular organization of hippocampus material stained with osmium - heavy metals .
however , we observed a marked increase in e - pta - stained material in subsevere pa .
although not too much data is available about the mechanism of cell death during pa [ 25 , 30 ] , these findings suggest that the increased in the thickness could be related with the degradation of abnormal proteins probably before neurons trigger death mechanisms .
thus , we hypothesize that some early signals triggered in psds could induce late neuronal alterations in post asphyctic hippocampal tissue . since we and others demonstrated that e - pta - stained aggregates could be composed of abnormal protein [ 25 , 33 , 46 ] , we performed immunoelectron microscopy following the procedures previously described by capani et al .   in order to detect ubiquitin ( figure 4 ) .
we observed ubiquitinated synaptic proteins after 19  min of pa in the stratum radiatum ca1 hippocampal area sections , while negative controls , in which the primary antibody was omitted , did not show immunolabeling ( data not shown ) .
taking these results into account , we could suggest that aggregates of ubi - proteins are present in psds of asphyctic animals , as it was observed in some neurodegenerative diseases . even though data about cell death mechanisms during pa are scarce [ 25 , 30 ] , these findings suggest that psd thickening could be related to degradation of abnormal proteins , probably before cell death mechanisms are triggered in neurons .
consistent with this view , persistent ubiquitination was found in the psd of hippocampal neurons [ 46 , 48 ] after transient cerebral ischemia , which suggests that increased ubi - protein conjugates might produce protein damage .
in addition , damage in protein can be produced by the increment in ros production and calpain activation as consequence of a rise in ca levels after hypoxic - ischemic insult [ 1 , 49 , 50 ] .
on the other hand , while other heat shock proteins reversibly attach to denatured proteins and help to refold or reassemble them , ubiquitin - conjugated proteins are degraded by 26  s proteasome .
pa insult activates the ubiquitin pathway , which might affect neuronal survival by damaged protein accumulation . since neurons
do not have the capacity to remove it , ubi - protein accumulation leads neurons to death .
brain hypoxia - ischemia triggers an early increment of glutamate in the extracellular space at synaptic level .
high levels of glutamate produce a cascade of events in dendritic spines that lead to cell death [ 25 , 31 , 33 , 43 , 48 , 5255 ] . since structure and function of dendritic spines are dynamically regulated by different cellular pathways acting on the actin cytoskeleton , we used light and electron microscopic techniques that had previously been used in our laboratory [ 1 , 56 ] and others [ 4 , 8 ] to study f - actin modifications induced by pa .
by confocal microscope analyses , we observed dendritic spines represented by punctate staining using phalloidin - alexa .
pa animals showed a decrease in punctate staining respect to ctl group ( figure 5  top ) ( p < 0.01 ) .
is mostly concentrated in mushroom dendritic spines , this decrement is tightly related with the f - actin contained in the dendritic spines .
when we analyzed different dendritic spine populations , we observed that only the number of mushroom dendritic spines , the only f - actin - positive spines in control animals , showed a significant decrease after 19  min of pa ( p < 0.05 ) ( figure 5  bottom ) .
in contrast , synapses did not show any sign of evident degeneration in asphyxic rats .
isolated synaptosome ( lp1 ) fractions were analyzed by immunoblotting using anti--actin antibody and quantified ( figures 5 and 6 ) .
statistical analysis showed no significant differences in mean optical densities of f - actin bands ( p = n.s . ) from pa and control group .
however , pa animals showed a decrease in amount of -actin with respect to ctl animals ( p = 0,058 ) .
both in vivo and in vitro studies showed high concentration of -actin in dendritic spines are involved in the organization of the synapses in adult brains [ 5760 ] .
although we observed a reduction of the number of spines f - actin positive in pa animals , the maintenance of -actin concentration observed in synaptosomal fraction may represent the cytoskeletal support of a stable dendritic spines structure that maintains , thus , the potential morphological plasticity in circumstances where adaptive changes in synaptic connectivity are adequate .
consistent with this point of view , disruption of receptor - scaffold proteins as nmdar - psd 95 , which depends on actin polymerization interactions , can prevent cell death after ischemia .
several studies using different models of neuronal cell death have demonstrated that endogenous gelsolin 's antiapoptotic properties correlated to its dynamic actions in the cytoskeleton .
gelsolin - null neurons have higher rates of cell death and a rapid and sustained elevation of ca levels following glucose / oxygen deprivation , as well as augmented cytosolic ca levels in nerve terminals following in vitro depolarization .
furthermore , the increment in histone acetylation induces upregulation of gelsolin , dramatically reducing the levels of actin filaments and cell death following cerebral ischemia in mice .
has shown that actin depolymerization prevents neuronal death , we hypothesize that the decrease in -actin in synaptosomal fraction could also be related with cell death observed after pa insult , as this process is connected with an abnormal ubi - protein increment .
in addition , our group has previously observed learning ; reference and working spatial memory impairments in the morris water maze in 3-months - old rat , subjected to acute asphyxia immediately after birth , using the hypoxic - ischemia model described in the present manuscript .
it is well known that the performance in these spatial tests is disrupted after hippocampal damage .
therefore , synaptic modifications observed in asphyctic animals could be related with behavioral deficits previously described by our group .
these findings suggest that excessive protein ubiquitination in hippocampal psd , 4 months after a subsevere pa insult , seems to be related to the increment in protein accumulation in this area . in spite of this increment
, we observed a decrease in -actin which suggests that pa is damaging the actin cytoskeleton .
moreover , the amount of -actin in pa animals is correlated with the decrement in the number of mushroom - shaped dendritic spines . although further studies will be necessary to determine the role of ubi - protein accumulation in psd , we could speculate that psd alterations might be involved in the generation of an aberrant biochemical pathway leading to long - term modifications in the brain of pa animals , as we described in a previous paper . in agreement with this point of view
, alzheimer 's disease has a deleterious action on the actin cytoskeleton linked with psd , leading to dendritic spine dysfunction and synaptic degeneration .
 perinatal asphyxia ( pa ) affects the synaptic function and morphological organization . in previous works 
 , we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia leading to long - term ubi - protein accumulation . 
 since f - actin is highly concentrated in dendritic spines , modifications in its organization could be related with alterations induced by hypoxia in the central nervous system ( cns ) . in the present study 
 , we investigate the effects of pa on the actin cytoskeleton of hippocampal postsynaptic densities ( psd ) in 4-month - old rats . 
 psd showed an increment in their thickness and in the level of ubiquitination . 
 correlative fluorescence - electron microscopy photooxidation showed a decrease in the number of f - actin - stained spines in hippocampal excitatory synapses subjected to pa . 
 although western blot analysis also showed a slight decrease in -actin in psd in pa animals , the difference was not significant . 
 taken together , this data suggests that long - term actin cytoskeleton might have role in psd alterations which would be a spread phenomenon induced by pa . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusions