inadequate blood flow to the brain , termed cerebral ischemia , will produce brain injury and a progression of pathophysiological responses associated with energy depletion that include cell swelling , cytotoxic edema , vasogenic edema and if sufficiently severe or prolonged , neuronal cell death .
when brain regions are directly damaged by ischemia this can lead to secondary injury or degeneration in associated axonal connections such as the corticospinal tracts .
detection of such changes on early subacute diffusion mri have been described as  early  or
such early signs of wallerian degenerative injury are predictive of specific functional outcomes and may improve prognostication , guide rehabilitation , and could represent both a selection criteria and potential novel target for clinical trials
. in particular , if there is diagnostic evidence for early degenerative injury , such as increases in the intensity of the corticospinal tract in diffusion weighted magnetic resonance ( mr ) images , then prognosis for a good recovery in such infants has consistently been poor ( domi et al . , 2009 ;
understanding better the cellular responses that evolve with axonal degeneration following stroke are important considering both their potential contributions to diagnosis and the potential for providing novel targets for therapeutic intervention .
currently , the tissue alterations underlying these mr biomarkers are unknown and speculative because histopathology , if available , is usually performed at single and relatively chronic time points many weeks to months after an ischemic insult
. studies of the evolution of ischemic brain damage or infarction at subacute and chronic times has been assessed using standard mr imaging sequences such as t2 or diffusion weighted imaging ( latchaw et al . , 2009 ) .
we recently demonstrated that following neonatal hypoxia with unilateral transient ischemia there is evidence for early wallerian degeneration visible as either decreases in the apparent diffusion coefficient ( adc ) and magnetization transfer ratio or increases in dw or t2 ( lama et al .
the early corticospinal axonal changes ( e.g. in the cerebral peduncle ) reflected several cellular alterations such as a reduced staining for phosphorylated neurofilament h and increased vacuolation in hematoxylin and eosin stained sections .
dti holds additional promise for detecting specific degenerative responses because of its potential sensitivity to certain ultrastructural cellular changes depending on the measured dti parameter  i.e. fractional anisotropy , mean diffusivity and parallel or radial diffusivity . of the extensive cellular responses produced by ischemia , those considered most likely to affect the diffusion properties of water are those that involve morphological tissue changes such as necrosis , astrogliosis , loss of myelin , loss of axonal neurofilaments or cellular inflammation ( i.e. microglial / macrophage activation ) ( nucifora et al . , 2007 ; zhang et al . , 2012 ) .
our group and others have shown that corticospinal tract dti alterations are strongly correlated with motor outcome in children with perinatal stroke ( hodge , 2013 ; roze et al .
however the progression of dti changes and whether there are differences between the evolution of cellular responses in direct ischemically injured brain versus associated connected tracts is not known .
differences are expected considering the different cellular compositions , for example neuronal rich cerebral cortex compared to axonal bundles within the descending corticospinal tract .
a differential timing of dti alterations is also expected considering direct ischemic injury is thought to be followed by a delayed wallerian degeneration ( lama et al . , 2011 ; tuor et al . , 2013 ) .
in order to better understand the unique cellular and dti imaging alterations associated with secondary wallerian degeneration relative to the onset and progression of direct ischemically damaged brain , it is necessary to compare their progression directly at multiple time points . in the current study we hypothesized that dti imaging would provide measures of progressive tissue changes within axonal tracts distal to the ischemic injury distinct from those in directly injured brain ( e.g. cerebral cortex ) and these differences would correspond with specific tissue morphological alterations in response to ischemia
this was investigated by using a neonatal rat model of unilateral transient cerebral hypoxia  ischemia and measuring dti changes in the parietal cerebral cortex compared with its associated descending corticospinal tract axon fibers within the cerebral peduncle .
dti measurements were made at acute , subacute and chronic times after hypoxia  ischemia along with processing of brains at each of these time points to assess immunohistochemically potential contributions of morphological modifications .
we focused on using markers for detecting cell death in neurons , loss of myelin and loss of neurofilaments in axons , and , activation of astrocytes and microglia / macrophages .
fifty one pups delivered from 9 different pregnant wistar female rats ( charles river laboratories , montreal , canada ) were used in the study .
experiments followed the canadian council on animal care guidelines and were approved by a university of calgary animal care committee .
a moderate unilateral ischemic lesion with hypoxia ( vannucci and vannucci , 2005 ) was produced as described previously ( lama et al .
briefly , pups ( n  =  38 ) on their 7th day of life had their right common carotid artery ligated under isoflurane anesthesia followed by a 60  minute exposure to hypoxia in a chamber containing 8% o2 and 92% n2 at 35.5  c .
sham control animals ( n  =  13 ) experienced a similar surgical procedure without carotid artery ligation and hypoxia exposure .
the vannucci model of neonatal cerebral hypoxia  ischemia ( vannucci and vannucci , 2005 ) produces ischemic damage and infarction within the distribution of the middle cerebral artery territory .
thus , the parietal cortex was selected as a representative brain region of direct hypoxic  ischemic damage or infarction .
the posterior cerebral peduncle supplied by the posterior circulation was selected as a region of secondary injury remote to the hypoxia 
brain near the aqueduct in the posterior pons was selected as a control region generally unaffected by the hypoxia  ischemia .
sham animals or animals subjected to cerebral hypoxia  ischemia were anesthetized ( 1.52% isoflurane ) and dti images in addition to anatomical scans were acquired at 3  h , 1  d , 2  d , 1  w or 4  w post - insult .
anatomical images were also acquired at 1  d post insult in the chronic animals ( 1 or 4  w ) to confirm the extent of ischemic damage .
mr images were acquired using a 9.4  t bruker biospin mr imaging system and paravision 5.1 software . throughout the scanning ,
respiration was monitored and maintained by adjustments in anesthesia and body temperature was maintained using a feedback heated air system ( small instruments inc . , stony brook , ny ) .
images were acquired using a 3.5  cm diameter quadrature volume coil for radiofrequency transmission and reception .
the head and body was restrained using custom designed swaddling and a head band or ear pins . depending on the age of the animal ,
each mr imaging scan consisted of 2530 slices of 0.50.55  mm thick covering the cerebrum and medulla , a 2    2  cm or 2.5    2.5  cm field of view and a data matrix size of 128    128 .
anatomical t2 maps were first generated using a t2 imaging sequence consisting of a set of t2 weighted spin echo images with 32 echoes , repetition time of 10  s and echo time of 10  ms between echoes .
for dti , a four - shot echo - planar imaging sequence was used to acquire four averages of sets of diffusion weighted images .
these were acquired with b values of 0 ( 5 images ) and 1000  s / mm ( 30 images in non - collinear directions ) using a repetition time of 6500  ms and an echo time of 35  ms . artifacts associated with imperfections in the radio frequency pulse , gradient stability , and gradient echo currents were removed using a navigator - echo phase correction .
nyquist ghost artifacts were suppressed in the image reconstruction using information acquired during the initial automatic receiver gain adjustment .
dti image acquisition time was approximately 1  h. t2 weighted images were visualized using local mr analysis software ( marevisi , national research council of canada , winnipeg , canada ) .
these images were used to assess the extent of ischemic damage and measure brain volumes ( atrophy ) .
inspection of t2 images was also used to exclude from further analysis animals with no cortical t2 lesions or very large hyperintense lesions extending into the pons .
they were also used to identify anatomical landmarks for selection of the regions of interest in subsequent dti analysis and histological analysis as identified using a rat brain atlas ( paxinos and watson , 1998 ) .
these regions of interest were manually defined in the mr images and histological sections and included the cerebral peduncle ( 0.30.4  mm ) , pons ( within the central gray and mesencephalic trigeminal nucleus regions , 0.60.9  mm ) and parietal cortex ( 0.71.0  mm ) for areas both ipsilateral and contralateral to the lesion containing hemisphere . once the ischemic infarct had progressed towards cavitation and cyst formation ( i.e. 1 and 4  w post - hypoxia  ischemia ) , homologous measures of cortical tissue were not possible and therefore were taken adjacent to the cyst  a peri - cyst cortical region ( 0.40.6  mm ) .
sizes and shapes were adjusted using the contralateral region ( e.g. cerebral peduncle ) as a guide and areas of interest were adjusted if there was atrophy in order to minimize partial volume effects .
diffusion tensor images were imported into dti studio ( jiang et al . , 2006 ) through the dti mapping function as philips rec files .
entire scans were excluded if less than eighteen artifact free images were available per slice .
the minimum of eighteen directions was selected empirically as the cut - off following an analysis of 12 test data sets with repeatedly fewer b directions ( e.g. 25 , 22 , 20 , 18 , 16 and 15 compared to 30 ) which demonstrated no significant effect of a reduction in the number of b directions except for fa and l3 ; and , those were altered only once the number of directions was 16 or less ( p  <  0.05 , repeated anova ) . for each image slice
the dti program calculated the over - determined matrix , the adc vector and the pseudo - inverse of the matrix using singular value decomposition in order to compute the tensor for each pixel ( jiang et al . , 2006 ) .
imaging maps were then calculated for the mean diffusivity ( adc ; ( 1  +  2  +  3 )  /  3 ) , the parallel diffusivity ( 1 ) , the radial diffusivity ( ( 2  +  3 )  /  2 ) and the fractional anisotropy ( fa ) . measures of the dti variables were obtained within regions of interest manually drawn ipsilateral and contralateral ( non - ischemic damaged side ) of the brain slice .
following their last mr scan , subgroups of animals were perfused transcardially with 10% formalin under deep pentobarbital anesthesia and their brains were embedded in paraffin .
brains from 44 rats were processed and stained for 5 different cellular or injury markers within sections containing the parietal cortex , posterior cerebral peduncle and central gray region of the pons .
thirteen of these animals were sham controls and 31 were animals subjected to hypoxia  ischemia and euthanized at the 5 different times post hypoxia  ischemia ( n  =  47/group ) .
sections ( 6  m thick ) were cut at the level of the posterior cerebral peduncle and parietal cortex using the brain atlas and anatomical landmarks to match sections to the mr slices and measure staining changes in equivalent regions of interest to those for the mr images .
sections from each region were stained for a marker of cell death or degenerating neurons using fluorojade b ( schmued and hopkins , 2000 ) according to the manufacturer 's instructions ( millipore , billerica , ma , usa ) . briefly , deparaffinized sections were exposed to 0.06% potassium permanganate for 15  min washed in distilled water and then stained with 0.0004% fluorojade solution .
sections at each level were also processed immunohistochemically to stain for the following : neurofilaments using a pan - axonal neurofilament marker ( monoclonal smi312 ) , reactive astrocytes using anti - glial fibrillary acidic protein ( gfap ) , activated microglia / macrophages using anti - cd68/ed1 , and , myelin using an antibody against myelin basic protein ( mbp ) .
sections were deparaffinized and processed for antigen retrieval by boiling in sodium citrate ( ph  =  6.0 , 90  c ) for 10  min then washed in pbs .
slides were subsequently blocked with 1% h2o2 in methanol for 5  min followed by 10% goat serum in 0.1  m pbs and then a pbs wash .
slides were then incubated for 1  h at room temperature with one of 4 different primary antibodies : smi312 antibody ( 1:1000 in bsa , covance , berkeley , ca , usa ) , polyclonal rabbit anti - gfap ( 1:500 in bsa , diagnostics canada inc .
, on , canada ) , mouse anti - rat cd68/ed1 ( 1:500 in bsa , abd serotec , raleigh , nc , usa ) or rabbit anti - mbp ( 1:500 in bsa , chemicon international , ca , usa ) . following a pbs wash
, slides were incubated overnight at 4  c with the appropriate secondary antibody , either biotinylated goat anti - rabbit igg or biotinylated goat anti - mouse igg ( 1:400 in bsa , jackson immunoresearch lab inc .
this was followed by incubation for half an hour with horseradish peroxidase - streptavidin ( 1:400 in pbs , diagnostics canada inc . , on , canada ) . finally , positive cells were visualized following an application of 3,3-diaminobenzidine ( sigma aldrich , mo , usa ) dissolved in 0.3% h2o2 in distilled water for approximately 3  min .
the histological sections were assessed semi - quantitatively for changes in staining within the regions of interest either by measuring optical density or by quantifying numbers of positively stained cells per field of view . for optical density measures , sections stained with either smi312 , gfap or mbp
were first captured digitally using a scanner ( nikon scan , version 4 , nikon inc . ) .
these images were converted to gray images and mean gray levels ( imagej , nih ) were measured within the same regions of interest as those drawn for the mr brain slices .
regions with less intense staining had higher mean gray values . the ipsilateral versus contralateral differences in gray levels were converted to a percentage of the contralateral value .
the cd68/ed1 and fluorojade stained sections were analyzed using digitized microscopy ( microbrightfield , olympus , 40 magnification ) .
the numbers of positively stained cells per field of view were estimated within regions corresponding to those selected from mr images .
three different fields of view were counted for positive staining cells by an investigator ( m.s . )
all group data is reported as mean    sd and statistical comparisons were considered significant at p  <  0.05 . measured values within different brain regions
a two way repeated analysis was used to determine significant region and time interactions and these were followed by a holm 
statistical tests were performed using sigmaplot ( systat software inc , san jose , ca ) . to convey clearly the differences for the cerebral peduncle , we noted in the graphs only the significant differences for this region .
the correlation between histological changes and dti parameters was assessed using a pearson product correlation analysis within each time point or all time points with a bonferroni adjustment for multiple comparisons .
despite intensive investigation of dti 's ability to detect brain injury , the interpretation of alterations in dti measures is usually speculative because of a lack of direct documentation of the corresponding histological changes . in the present study
we report detailed differences in the progression and magnitude of dti and histological changes that provide a reference for identifying the potential contribution of various cellular responses to fa and axial , radial and mean diffusivity .
the results are first to characterize the correspondence of the progression of cellular and dti changes within the cerebral peduncle of the descending corticospinal tract following hypoxic  ischemic damage to the cortex .
novel is the demonstration of the differing progression of dti alterations in a directly damaged ischemic region such as parietal cortex versus that in associated subcortical tracts .
dti changes were less severe or delayed in the cerebral peduncle of the corticospinal tract and this in general corresponded to less severe and delayed cellular responses consistent with early wallerian degeneration .
as discussed in more detail , the tissue alterations following ischemia and axonal degeneration had a complex relationship with changes in fa , parallel diffusivity and radial diffusivity .
regarding our assessment of progressive changes in the dti parameters , it is important to realize that superimposed on the effects of hi injury are well recognized developmental changes in the dti parameters that occur during development and aging ( dubois et al . , 2013 ) .
indeed , the contralateral hemisphere was similar to those in sham animals but developmental changes may have been differentially affected in the left and right hemispheres by the ischemic insult .
right differences needs to include an awareness that these can be accounted for by an injury modulation of cellular maturation processes in white matter , which we assessed in our histological sections .
the region of cerebral peduncle measured was approx 0.30.4  mm consisting of approx 10 pixels of sizes 0.024  mm in the neonatal scans and 0.038  mm in 5  wk old animals .
partial volume effects could be greater in neonatal brain than older brain . however , because we compared left and right differences in values , we expect that there were similar ipsilateral and contralateral partial volume effects in the cerebral peduncle at each time point .
right differences but this did not appear to be an issue considering that many of our measures had the maximal changes in our neonates ( e.g. 1  d post insult ) . there have been numerous studies regarding the evolution of adc or mean diffusivity following transient cerebral ischemia in regions of stroke .
adc alterations reported previously are comparable to those observed presently ( e.g. lama et al . , 2011 ; liu et al . , 2007 ) .
notably , there were striking similarities between the adc and parallel diffusivity changes suggesting that both reflect similar cellular responses to injury . very early
adc and parallel diffusivity changes in ischemic cortex are likely due to a greater restriction of water diffusion as a result of cytotoxic edema , reduced extracellular space and increased tortuosity which in the present study was also accompanied by decreased neurofilament staining and increased neuronal cell death . as vasogenic edema and damage to cellular membranes proceeded , which histologically corresponded to a continuing cell death and microglial / macrophage activation and astrogliosis , the reductions in adc and parallel diffusivity normalized and this process occurred over several days .
by 14  w , phagocytosis produced cysts in the infarct region and such cystic regions had an extremely large adc in all directions whereas peri - infarct regions generally had normalized adc and normalized parallel diffusivity despite the presence of inflammatory cells .
in contrast , in the cerebral peduncle with indirect ischemic injury , by the first 3  h there were modest but significant adc and parallel diffusivity reductions .
the hyperacute changes in adc and parallel diffusivity correspond to our previous observation of a reduction in smi31 , demonstrating a decrease in phosphorylated heavy neurofilament subunits already hours after insult .
subsequently , the adc and parallel diffusivity reductions increased in magnitude and were maximal in the first few days .
this corresponded well with the diminished staining observed for the pan - axonal ( heavy , medium and light ) neurofilament marker smi312 used presently .
loss of phosphorylation of neurofilaments promotes their disassembly and this corresponded to a decreased radial diffusivity as well as a decreased parallel and mean diffusivity of water . at chronic time points post - insult ( 14  w ) ,
a similar greater sensitivity for parallel diffusivity than adc in detecting wallerian degenerative changes in the corticospinal tract has been reported after an excision lesion of the feline cortex ( qin et al . , 2012 ) .
microglial / macrophage cell activation likely did not contribute greatly to the parallel diffusivity alterations observed , considering the rather modest positive ed1/cd68 staining observed in the cerebral peduncle .
the occurrence of astrogliosis coincided with normalization of adc potentially contributing to some extent to these changes .
however , as discussed below , there were also ipsilateral increases in radial diffusivity that likely contributed to the adc normalization .
reductions in radial diffusivity have been proposed to reflect well increases in myelin whereas elevations in radial diffusivity have been found to be associated with a loss of myelin ( song et al . , 2002 , 2004 ;
the current study provides some support for this if one examines only chronic recovery times  i.e. times when the axons in the contralateral cerebral peduncle have become well myelinated . at 1  w and beyond , myelin within the cerebral peduncle
ipsilaterally was reduced reflecting less ontogenic myelination of fewer axons following their degeneration and atrophy .
this coincided with relative increases in radial diffusivity ipsilaterally and is consistent with increases in radial diffusivity reported in the corticospinal or pyramidal tracts at relatively chronic times ( e.g. 1  w to 3  y ) following an ischemic insult in children or adults ( roze et al .
, 2012 ; yu et al . , 2009 ; zanier et al . , 2013 ) . at acute times post - insult we observed ipsilateral reductions in radial diffusivity within the cerebral peduncle indicating that in the absence of effects on myelination , early axonal injury such as that detected by decreases in phosphorylated neurofilaments were also associated with reduced radial diffusivity
it is noteworthy that the neurophysiological correlates of alterations in parallel and radial diffusivity are potentially more complex in humans than rodents .
a diffusion tensor analysis has an implicit assumption that the white matter fibers are cylindrical and when fibers have complicated microstructures or crossing fibers as occurs in human developing brain ( dubois et al . , 2013 ;
2013 ) then in particular the directions of orientation for tractography require careful interpretation . despite crossing fibers potentially complicating the interpretation of parallel diffusivity changes ,
transverse or radial diffusivity appears to consistently decrease with all maturational processes within white matter , including myelination ( dubois et al . , 2013 ) .
furthermore , emerging imaging methods exploring diffusion parameters beyond the tensor model may provide more in depth assessments to facilitate translation from animals to humans ( dubois et al .
the fa measures provided a sensitive measure of direct ischemic injury . in the cortex at hyperacute times
, there was an increase in fa , as has been observed previously in rats and non - human primates , however , usually soon after permanent rather than transient occlusion ( kim et al .
, 2013 ; liu et al . , 2007 ; yang et al . , 1999 ) .
this suggests that the very early cortical ischemic changes were relatively severe and similar to those with permanent ischemia .
cellular changes likely involve previously established hyperacute cytotoxic cell swelling and reductions in extracellular space leading to cell death considering our histological evidence for hyperacute neuronal cell death in the cortex . by the first day post - insult ,
the fa increase became a decrease that persisted at subacute and chronic time points , confirming previous reports of a reduced fa commonly observed in damaged brain post - stroke ( e.g. kim et al .
, 2013 ; liu et al . , 2007 ; yang et al . , 1999 ) .
this decrease in fa , reflecting diminished preferential diffusivity , is likely associated with a loss of membrane integrity and intracellular structure due to necrosis as supported histologically by continuing cell death and loss of neurofilament staining .
the increase in astrogliosis between 3 and 1  d appears rather isotropic considering that there is a decline in fa between these times .
the fa also provided a rather robust measure of degenerative changes in the cerebral peduncle .
in contrast to ischemic cortex , fa in cerebral peduncle was decreased at all time points .
indeed , fa appeared to be the most sensitive dti parameter for detecting axonal degenerative changes in the cerebral peduncle independent of time post - insult and this corresponded well to the persistent decreases in axonal neurofilament staining .
similar reductions in fa have been reported at various select times in the corticospinal tract following neonatal stroke ( ludeman et al . , 2008 ; puig et al . , 2013 ; tusor et al . , 2012 ) .
it is currently clear that at chronic times , fa is superior to adc or other standard imaging methods such as t2 imaging because lesions detected with these other sequences tended to pseudo - normalize at times post - insult ( lama et al . , 2011 ; tuor et al . ,
the results demonstrate that in general , diffusivity changes and histological responses within directly damaged ischemic brain precede and are often larger in magnitude than corresponding alterations in a region of secondary axonal degeneration , such as the cerebral peduncle . with the progression of damage over one or more weeks , dti components such as fa and radial diffusivity are better than mean adc for detecting ischemia associated axonal injury indicative of wallerian degeneration .
also , differences in radial diffusivity in brain injured perinatally are likely related to a lack of ontogenic myelination on the injured side .
thus , the specific progression of dti alterations following an ischemic insult not only depends on the maturity of the subject but also appears dependent on other factors such as the type of insult ( e.g. spinal cord injury or brain resection ) ( brennan et al . , 2013 ; liu et al . , 2013 ; qin et al . , 2012 ) .
the inflammatory response investigated ( microglial or astroglial activation ) appears to have modest contributions to the evolution of axonal changes in diffusion tensor parameters .
thus , this systematic study is informative regarding the optimal dti mr imaging parameters to probe axonal injury following hypoxic / ischemic brain damage in developing brain in addition to aiding our interpretation of dti parameters for detecting various stages of wallerian degeneration .
 ischemically damaged brain can be accompanied by secondary degeneration of associated axonal connections e.g. wallerian degeneration . diffusion tensor imaging ( dti ) 
 is widely used to investigate axonal injury but the cellular correlates of many of the degenerative changes remain speculative . 
 we investigated the relationship of dti of directly damaged cerebral cortex and secondary axonal degeneration in the cerebral peduncle with cellular alterations in pan - axonal neurofilament staining , myelination , reactive astrocytes , activation of microglia / macrophages and neuronal cell death . 
 dti measures ( axial , radial and mean diffusivity , and fractional anisotropy ( fa ) ) were acquired at hyperacute ( 3  h ) , acute ( 1 and 2  d ) and chronic ( 1 and 4  week ) times after transient cerebral hypoxia with unilateral ischemia in neonatal rats . the tissue pathology underlying ischemic and degenerative responses had a complex relationship with dti parameters . 
 dti changes at hyperacute and subacute times were smaller in magnitude and tended to be transient and/or delayed in cerebral peduncle compared to cerebral cortex . in cerebral peduncle by 1  d post - insult , there were reductions in neurofilament staining corresponding with decreases in parallel diffusivity which were more sensitive than mean diffusivity in detecting axonal changes . 
 ipsilesional reductions in fa within cerebral peduncle were robust in detecting both early and chronic degenerative responses . at one or four weeks post - insult , radial diffusivity was increased ipsilaterally in the cerebral peduncle corresponding to pathological evidence of a lack of ontogenic myelination in this region . 
 the detailed differences in progression and magnitude of dti and histological changes reported provide a reference for identifying the potential contribution of various cellular responses to fa , and , parallel , radial , and mean diffusivity . 
Introduction
Material and methods
Discussion