Recurrent mild cerebral ischemia: enhanced brain injury following acute compared to subacute recurrence in the rat

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1 DOI / x BMC Neurocience RESEARCH ARTICLE Open Acce Recurrent mild cerebral ichemia: enhanced brain injury following acute compared to ubacute recurrence in the rat Urula I. Tuor 1,2*, Zonghang Zhao 1, Philip A. Barber 1 and Min Qiao 2 Abtract Background: In the current tudy, a tranient cerebral ichemia producing elective cell death wa deignated a mild ichemic inult. A comparable inult in human i a tranient ichemic attack (TIA) that i aociated with functional recovery but can have imaging evidence of minor ichemic damage including cerebral atrophy. A TIA alo predict a high rik for early recurrence of a troke or TIA and thu multiple ichemic inult are not uncommon. Not well undertood i what the effect of differing recovery time between mild ichemic inult ha on their pathophyiology. We invetigated whether cumulative brain damage would differ if recurrence of a mild ichemic inult occurred at 1 or 3 day after a firt inult. Reult: A tranient epiode of middle cerebral artery occluion via microclip wa produced to elicit mild ichemic change predominantly cattered necroi. Thi wa followed 1 or 3 day later by a repeat of the ame inult. Brain damage aeed hitologically 7 day later wa ubtantially greater in the 1 day recurrent group than the 3 day recurrent group, with area of damage coniting predominantly of region of incomplete infarction and pannecroi in the 1 day group but predominantly region of elective necroi and maller area of incomplete infarction in the 3 day group (P <.5). Enhanced injury wa reflected by greater number of cell taining for macrophage/microglia with ED1 and greater alteration in GFAP taining of reactive atrocyte in the 1 day than 3 day recurrent group. The differential uceptibility to injury did not correpond to higher level of injuriou factor preent at the time of the econd inult uch a BBB diruption or increaed cytokine (tumor necroi factor). Microglial activation, with potential for ome beneficial effect, appeared greater at 3 day than 1 day. Alo blood analyi demontrated change that included an acute increae in granulocyte and decreae in platelet at 1 day compared to 3 day pot tranient ichemia. Concluion: Dynamic change in multiple inflammatory repone likely contribute to the time dependence of the extent of damage produced by recurrent mild ichemic inult. The time of mild troke recurrence i crucial with early recurrence producing greater damage than ubacute recurrence and thi upport urgency for determining and implementing optimal troke management directly after a TIA. Keyword: Cerebral ichemia, Tranient ichemic attack, Stroke, Recurrence, Inflammation, Granulocyte Background Ichemic troke remain a leading caue of death and a major caue of adult diability reulting in a huge peronal, *Correpondence: utuor@ucalgary.ca 1 Department of Clinical Neurocience and Hotchki Brain Intitute, Cumming School of Medicine, Univerity of Calgary, Calgary, AB T2N 4N1, Canada Full lit of author information i available at the end of the article ocial and economic burden [1]. Often conidered a warning ign for troke, a tranient ichemic attack (TIA) reult from a cerebral ichemic epiode of hort duration aociated with a temporary blockage of a cerebral artery reulting in tranient (le than 24 h) functional deficit [2, 3]. Although TIA are aociated with ubtantial or full functional recovery, there i both experimental and clinical evidence that reperfued brain may often uffer ome 216 The Author(). Thi article i ditributed under the term of the Creative Common Attribution 4. International Licene ( which permit unretricted ue, ditribution, and reproduction in any medium, provided you give appropriate credit to the original author() and the ource, provide a link to the Creative Common licene, and indicate if change were made. The Creative Common Public Domain Dedication waiver ( publicdomain/zero/1./) applie to the data made available in thi article, unle otherwie tated.

2 Page 2 of 14 permanent damage uch a cattered cell death or elective neuronal lo following a tranient ichemic event [4 7]. Undertanding better the pathophyiology of uch mild tranient ichemic injury i of ome urgency conidering an anticipated higher incidence of TIA a the population become increaingly elderly, and, an anticipated increaing number of tranient cerebral artery occluion will be uccefully recanalized with tiue plaminogen activator or endovacular therapy [8 1]. In addition to being key to treatment approache, improved knowledge regarding the pathophyiology of a relatively hort tranient ichemic inult reulting in recovery i alo of importance for undertanding the interaction of multiple event conidering that a TIA i aociated with an increaed rik of a econd or recurrent troke [2, 3, 11]. A majority of TIA are aociated with cortical ichemic event and large veel dieae and recurrence i mot commonly aociated with a econd ipilateral cortical ichemic event [12, 13]. However, urpriingly little i known of the interaction of the ichemic injury procee that occur with multiple mild ichemic inult where the inult are ufficiently evere to caue elective cell death within the ichemic territory. Duration between inult i likely a crucial factor conidering that ichemic injury and brain recovery evolve over time. Furthermore, the pathophyiology of early recurrence i important to undertand conidering recurrence i frequently oberved by 1 day after a TIA [14]. Thu the objective of the current tudy wa to determine whether cumulative damage from multiple mild tranient ichemic inult, each of ufficient everity to caue elective necroi, i influenced by the recovery time between inult. A econdary objective wa to determine whether the evolution of cellular change after the firt inult were aociated with the total damage produced by the recurrent inult. We hypotheized that damage with an early recurrent troke would be greater than with a ubacute recurrent troke. An animal model wa conidered key becaue ichemic everity could be varied to produce mild ichemic cerebral change a can be oberved clinically. Furthermore the timing between inult could be well controlled. Thu we ued a rat model of tranient focal ichemia [7, 15] adjuted to produce elective cell death, which in young Witar rat required 3 min of occluion; thi wa deignated to be a mild tranient cerebral ichemic inult. We alo produced a recurrence of the inult at 1 or 3 day with the reult demontrating that brain damage i greater when recurrence i acute (1 day) rather than ubacute (3 day). Method Male Witar rat (Charle River, Montreal, Canada) were acclimatized to a 12 h light/dark cycle with free acce to food and water. Animal were randomized to one of three group involving double urgical procedure: a control group with ham urgery + tranient middle cerebral artery occluion () at 1 day and experimental group with + recurrent at 1 day and + recurrent at 3 day (n = 6/ group). Sample ize wa elected to minimize number of animal ued yet provide power a etimated to detect a 3 % difference between the anticipated mean of group auming a tandard deviation of 15 %. Animal were euthanized 7 day after the lat urgery and brain were proceed for hitology to determine the extent of ichemic brain damage. In additional experiment performed to invetigate longitudinal cerebral ichemic change, animal were randomized to and euthanaia at 1 or 3 day after the tranient ichemia (n = 8/group). Peripheral ytemic inflammatory change were alo invetigated in additional animal from blood ampled at either 1 or 3 day following tranient (n = 5/group). Tranient mild focal ichemic inult Surgical procedure were performed aeptically under ioflurane anetheia and included analgeic meaure to alleviate uffering. On the day of the firt urgery, a tranient mild ichemic inult (mild tranient ) wa produced in the laboratory by temporarily occluding the dital MCA uing a microurgical approach a decribed previouly [7, 15]. Briefly, a microaneurym clip wa placed on the MCA through a mall burr hole in the temporal bone above the MCA where it croe the rhinal fiure. Rectal temperature wa maintained uing a ervocontrolled heating lamp. A tail artery wa cannulated for obtaining arterial blood ample and a mall burr hole over the ipilateral parietal cortex wa ued to meaure cerebral perfuion with laer Doppler flowmetry. Concurrent to the, both common carotid arterie were traniently occluded uing uture thread. At the end of the 3 min occluion, the microclip and carotid artery ligature were removed. A dura ubtitute (Gore Preclude MVP, Better Hopital Supplie Corp., Miami, FL) wa poitioned on cloing to facilitate the production of a ubequent econd. Topical anethetic in addition to the adminitration of buprenorphine (.3 mg/ kg,.c. every 12 h a needed) provided analgeia. During a ham operation, animal underwent the ame urgical procedure with the exception of vacular occluion. Following urgery, rat were houed in eparate cage with free acce to oft and hard food, water and environmental enrichment. Animal were monitored twice daily for the firt 2 day and then daily to enure good recovery. Animal were ubjected to a recurrent mild focal ichemic inult by repeating the 3 min procedure at either 1 or 3 day of recovery following the initial

3 Page 3 of 14 mild. The location of the econd clip placement on the MCA wa immediately dital to the original poition to minimize urgical complication. Hitology to detect brain injury or repair repone For hitological analyi, pentobarbital anethetized rat were perfuion fixed with formalin and brain were embedded in paraffin. Section (6 μm) were tained with tandard and immunohitochemical method and altered taining or ichemic injury were aeed blinded to the animal identification or urgical group. Standard taining with hematoxylin and eoin In ection tained with hematoxylin and eoin, the extent of cortical damage wa identified by claifying ichemic injury in the ichemic middle cerebral artery territory a elective necroi, incomplete infarction or pannecroi a decribed in previou tudie [5, 16]. In addition, a core for brain injury wa determined [7] by dividing the cortex into 4 region of interet and a core wa aigned to each region graded a: for normal, 1 for <1 % of cellular injury, 2 for 1 5 % of cellular injury, 3 for >5 % cellular injury and, 4 for confluent area of pannecroi. A cumulative core wa obtained by umming the core for each region. Immunohitochemical taining for cellular ichemic change Macrophage/microglia and atrocyte are two glial cell type participating in the CNS inflammatory repone to cerebral ichemia. The ichemic change in thee cell oberved following 7 day of recovery after multiple ichemic inult alo reflect the everity of ichemic injury. Reactive atrocyte were aeed uing a primary antibody to glial fibrillary acidic protein (GFAP) (rabbit anti-gfap, 1:1,, AB_113482, Dako, Burlington, ON). Increaed activation of macrophage/microglia were aeed uing anti-ed1 (AB_22913, moue anti-rat CD68 clone ED1, 1:1, AbD Serotec, Raleigh, NC) [17]. The effect of a ingle mild ichemic inult on the progreion of inflammatory change between 1 and 3 day were aeed firt by taining for reactive atrocyte and macrophage/microglia a above. Change in the cytokine, tumor necroi factor (TNF) wa aeed uing goat anti rat TNF-alpha (1:1, AB_354511, R&D ytem, Minneapoli, MN) [17]. In addition, early activation of microglia wa aeed uing an antibody againt Ionized Calcium-Binding Adapter Molecule 1 (Iba1, 1:1, AB_83954, Wako Chemical, USA) which i pecifically expreed in microglia and i upregulated following cerebral ichemia [18]. Vacular change were invetigated uing a fluorecein iothiocyanate-labeled tomato lectin (1:2, AB_23744, Vector Laboratorie Burlingame, CA) that tain both microglia and veel [19]. The ame ection tained for lectin were alo tained uing a primary moue anti-rat endothelial barrier antigen (EBA) IgM (AB_11265, SMI 71, 1:4, Covance) that tained veel [2] to ae blood brain barrier dyfunction. Comparing EBA and lectin tained ection provided an additional detection of morphological and intenity taining change in microglia. Finally, integrity of the blood brain barrier wa aeed uing an antibody to detect extravaation of large plama protein (1:2, goat anti rat immunoglobulin G (IgG), Jackon ImmunoReearch Lab, Wet Grove, PA). For all the immunohitochemical tain, method were generally a decribed previouly [7]. Briefly, paraffin ection were firt proceed for antigen retrieval uing citrate buffer at ph = 6, an exception being ection tained with Iba1. The ection were incubated with either 1 % goat or donkey erum, then primary antibody, followed by the appropriate biotin-conjugated IgG (Jackon ImmunoReearch Lab, Wet Grove, PA) or fluorecent conjugated econdary antibody. For colorimetric taining, horeradih peroxidae conjugated treptavidin (1:4, Dako, Burlington, ON) and diaminobenzidine (Sigma) were applied. For double taining of lectin and EBA, antigen retrieval wa omitted and after blocking with goat erum ection were incubated with FITC-lectin at room temperature for 1 h followed by incubation with EBA at room temperature overnight. Hitological analyi of altered immunotaining Section were viualized and analyzed blinded to the experimental group uing an Olympu BX61 microcope and Microbrightfield Stereo Invetigator (MBF Biocience, Williton, VT). Immunohitochemical change were emi-quantified according to the tain ued and the type of cellular change oberved in the MCA territory of the parietal cortex ipilateral and contralateral to the tranient occluion. For ED1 and TNF, region of parietal cortex within the MCA territory were inpected and cell within at leat three field of view (.263 mm 2 each) with poitive taining for ED1 or TNF were counted and ued to provide a mean value. For EBA, fluorecent image of parietal cortex were captured digitally (2 magnification) and thoe with technically good veel taining in the contralateral hemiphere were analyzed uing Image J for counting of the number of EBA tained veel in repreentative ipilateral and contralateral field of view. GFAP tained ection were aigned cumulative core imilar to the hematoxylin and eoin tained ection, with altered level of taining being cored in each of the four

4 Page 4 of 14 different cortical area a: for normal, 1 for cattered reactive cell labelled or <1 % of area, 2 for 1 5 % of the area labelled, 3 for >5 % of the area labelled and 4 for confluent area of pannecroi with total lo of GFAP tain. A lectin taining core wa attained by aeing the extent of increaed tomato lectin taining of activated microglia and their procee within the cortical parenchyma (i.e. in exce of veel labelled with EBA) and cored according to: none, 1 pare number and 2 ubtantial number of parenchymal cell with poitive lectin taining. In region of infarct a core of 3 wa aigned if there were ubtantial number of poitive tained parenchymal cell in addition to a diffue increae in ignal from within the parenchyma. For Iba1 tained ection, thoe with the bet poitive taining of procee were elected for counting both the total number of microglial cell tained per field and the number of activated microglial cell with ramified or thickened buhy procee. Potential increaed colorimetric taining in cortex for plama protein with IgG were aeed by meauring gray level in ipilateral and contralateral cortex along with level on the blank lide adjacent to the region of interet in order to analyze left right difference. Sytemic circulatory repone Hematology analyi of blood ample wa alo performed in additional rat to ae for ign of immunouppreion or infection. Venou blood ample (.2 ml) were collected in EDTA coated tube and analyzed immediately for concentration of platelet and red and white blood cell (HemaTrue Hematology Analyzer, Heka Corp., Loveland, CO, USA). Relative proportion of the different type of white cell were alo aeed by meauring the number of monocyte, granulocyte and lymphocyte. Statitical analyi Data were analyzed uing SigmaPlot 13 oftware (Sytat Software Inc, San Joe, CA). Data are reported a mean ± SD for group with continuou value or a the median and firt and third quartile for nonparametric data. Statitical comparion were conidered ignificant at P <.5. Prior to a comparion of mean, ample were firt teted for normality and equal variance followed by election of an appropriate analyi of variance and pot hoc tet (e.g. Bonferroni). Difference between ipilateral and contralateral hemiphere were compared uing a paired Student t-tet or a Rank Sum Tet for categorical value. Reult Brain damage varie with timing between multiple mild inult For animal receiving multiple urgical intervention, eight animal were lot to analyi due to mortality pot recurrent troke (n = 1), inadequate flow reduction during clip placement (n = 2), urgical complication (n = 2) and a problem with tiue embedding (n = 2). A total of 18 animal (n = 6/group) were analyzed in the ham + mild, 1 day recurrent and 3 day recurrent group. Body weight in thee 3 group were imilar at baeline and time of the econd urgery (29 ± 65 and 282 ± 5; 319 ± 56 and 314 ± 65; 296 ± 45 and 296 ± 4, repectively). In all animal, ichemic change were oberved in the cerebral cortex ipilateral to the tranient middle cerebral artery occluion (Fig. 1b, d, e h) but not in the contralateral cortex (Fig. 1a, c). Severity of injury varied between group (ee alo data in Additional file 1). Subtantial area of incomplete infarct and pannecroi in addition to ome area of elective necroi were apparent within the middle cerebral artery territory in all 6 animal ubjected to a recurrent mild ichemic inult eparated by 1 day (e.g. Fig. 1b, d). Subtantial change in ED1 and GFAP taining alo occurred with recurrence at 1 day pot firt inult (Fig. 1j, l and o, q, repectively). In contrat, in animal ubjected to a recurrent mild ichemic inult eparated by 3 day there were only mall region of incomplete infarction with more extenive region of elective necroi in four of the ix animal and only region of elective necroi in 2 animal (e.g. Fig. 1e, g). Poitive taining of macrophage/microglia with ED1 or for reactive atrocyte with GFAP wa alo modet (Fig. 1k, m, p, r). Brain of animal that underwent a ham procedure followed by tranient ichemia 1 day later had the leat extenive ichemic change coniting of only elective necroi (6/6 animal) (e.g. Fig. 1f, h) and minimal taining for ED1 or GFAP (3/3) (Additional file 1). Semi-quantitative analyi of the tained ection upported thee obervation (Fig. 1i). All group had ignificantly greater damage (median cumulative core 3) in the ichemic cortex than in the contralateral hemiphere which wa normal (median core of ). Animal with an early recurrent troke 1 day after the firt inult had ignificantly greater damage aeed in hematoxylin and eoin tained ection than a ham procedure followed by a mild. Total damage with early (1 day) recurrence alo exceeded that produced by recurrent troke eparated by 3 day (P <.5). The difference in everity of damage and their dependence on the recovery time between the firt and recurrent inult were alo reflected in an increaed number of cell taining with ED1

5 Page 5 of 14 a b c Contralateral d x2 1 d i p p Contralateral e x2 3d p g x2 3d Sham+ l x2 m x2 3d x2 x2 3d o p q x2 r p p x2 j x2 Sham+ x2 3d f k p h x2 3d 25 µm Ipilateral H&E (Score) n Ipilateral ED1 (Cell/Field) Ipilateral GFAP (Score) d Recovery Before Recurrent * ** 3d Recovery Before Recurrent * * 3d Recovery between Procedure Fig. 1 Cerebral injury dependence on recovery time between recurrence of mild tranient ichemic inult. Repreentative cerebral cortical micrograph of ection at low and high magnification (cale bar of 25 µm) tained with: hematoxylin and eoin (a h), ED1 for activated macrophage/ microglia (j m), and GFAP for reactive atrocyte (o r). Brain wa perfuion fixed and proceed 7 day following the lat inult. Cerebral cortex contralateral to the tranient middle cerebral artery occluion () i normal (A,C). Within cerebral cortex (b, d) after a mild tranient followed by a recurrent at 1 day there i extenive pannecroi (p). Selective necroi () i common following a mild tranient followed by a recurrent at 3 day (e, g). Selective necroi alo occur in rat with a ham urgery followed 1 day later by tranient (f, h). Semiquantitative aement of injury core indicated greater damage with 1 day compared to 3 day recurrence in the H&E ection (i). Immunohitochemical taining with ED1 antibody demontrated an increae in activated macrophage/microglia following recurrent produced at either 1 or 3 day after an initial (j m). The mean number of cell per field with ED1 taining demontrating a difference between 1 and 3 day recurrent group (n). GFAP taining of atrocyte wa increaed with recurrent at 1 or 3 day (o r). The median core for altered GFAP taining reflected greater injury and atrocytic change in brain of animal with recurrent at 1 day than 3 day (). n = 6/group. *P <.5; **P <.6, Ipilateral different from contralateral; P <.5; P <.6, different from 1 day Recurrent. P <.1, different from Sham ** ** ** (Fig. 1n) and the everity core for difference in taining for reactive atrocyte with GFAP (Fig. 1). Similar phyiological meaure in recurrent troke group Potential difference in everity of ichemia or other meaured phyiological parameter that are well known to affect ichemic injury were comparable between group. Cortical perfuion wa le than 1 % baeline during and reperfuion level were imilar during both the firt and econd ichemic inult irrepective of the recovery time between them (Fig. 2a). There wa alo excellent control of body temperature during and during early reperfuion, irrepective of the experimental group (Fig. 2b). Other phyiological variable and blood gae were imilar between 1 and 3 day recurrent group. Mean value were 86 ± 11 and 88 ± 11 mm Hg for mean arterial blood preure, 12 ± 7 and 19 ± 5 for PO2, 44.2 ± 2 and 38.6 ± 2 mm Hg for PCO2, 7.35 ±.1 and 7.36 ±.4 for PH and 13.6 ± 3 and 11.6 ± 2 mmol/l for blood glucoe, repectively. See alo data in Additional file 2. Inflammatory change following a ingle mild tranient ichemia In order to invetigate whether differential repone to brain injury and recovery from ichemia could explain the enuing damage produced by a recurrent, longitudinal tiue change to a ingle mild were

6 Page 6 of 14 a Cortical perfuion (% Baeline) b Rectal Temperature ( C) d During Pot During Pot 1 t 2 nd 3 d During Pot During Pot 1 t 2 nd Fig. 2 Mean cortical perfuion or rectal temperature during or pot middle cerebral artery occluion (). a Cortical perfuion meaured uing Doppler flowmetry preented a a percent of baeline. Mean perfuion decreaed to <8 % baeline during ubequently returning toward baeline within the firt 5 min following reperfuion irrepective of recurrence at either 1 or 3 day pot the firt (n = 6/group). b Mean core body temperature wa well controlled reulting in imilar mean for the recurrent group either during or after the firt or econd invetigated in additional randomized animal. Several of thee were lot to analyi due to mortality (n = 1), urgical complication (n = 2) and a problem with tiue embedding (n = 1). Section from all animal analyzed following a ingle tranient demontrated mild damage in hematoxylin and eoin tained ection (Fig. 3a, b). Damage core wa imilar in the 1 and 3 day recurrent group (n = 8/group) (Fig. 3c). In adjacent tained ection, there were increaed number of ED1 tained macrophage/microglia ipilaterally in both group (Fig. 3d, e). There wa alo altered GFAP taining of reactive atrocyte (Fig. 3g, h) compared to contralateral cortex. Thee inflammatory change cored in the 1 and 3 day recurrent group were not different tatitically (Fig. 3f, i). See alo data in Additional file 3. In ection from thee animal we alo invetigated whether TNF-alpha, often conidered to be a pro-inflammatory cytokine, i ubtantially increaed 1 day pot a mild ichemic inult and thereby a potential contributor to increaed ichemic damage oberved with multiple inult at thi time. Relative to the contralateral cortex, which had minimal TNF taining (Fig. 4a), increaed number of poitive TNF tained cell were oberved ipilaterally at 1 and 3 day pot-inult (e.g. Fig. 4b, c). The mean increaed number of TNF tained cell were not different tatitically at 1 and 3 day pot inult (Fig. 4d). See alo data in Additional file 4. Microglial change, which are uually conidered proinflammatory acutely but can alo contribute later to repair [21], were altered following a ingle mild inult. Microglial cell tained with Iba1 (Fig. 4e h) were imilar in the 1 and 3 day group contralaterally (e.g. mean of 23. ± 6. cell per field). Total number of cell ipilaterally increaed to 32.4 ± 9 and 64.9 ± 25 in the 1 and 3 day group repectively (P <.5) and included cell with ramified or buhy morphology or round cell (Fig. 4f, g). Thoe with ramified or buhy morphology were alo different between group (Fig. 4h). See alo data in Additional file 4. Uing a tomato lectin tain of veel and microglia, examination of taining in the non-vacular parenchyma contralaterally indicated a lack of appreciable taining for microglial cell with lectin [22] (e.g. Fig. 4i). At 1 day pot there wa a modet increae in lectin tained microglia and their procee, aociated with microglial activation (e.g. Fig. 4j). At 3 day pot-inult, ubtantial number of microglia tained for lectin (e.g. Fig. 4k) and the median core for lectin taining differed ignificantly between group (P <.5) (Fig. 4l). BBB injury following a mild tranient ichemia In ection tained with lectin, we alo performed immunotaining for endothelial barrier antigen (EBA) whoe lo i conidered to provide a highly enitive indicator of dyfunction of the blood brain barrier [2]. There wa reduced vacular EBA immunoreactivity oberved following a mild ichemic inult. Compared to the contralateral hemiphere (Fig. 4m), there were fewer EBA tained veel (Fig. 4n o) and analyi of ection determined a reduced number of EBA tained veel both at 1 and 3 day pot a mild (Fig. 4p). There wa no ignificant difference between group. Depite a reduction in EBA taining there wa no evidence of major BBB damage. Following a ingle mild ichemic inult, there wa a lack of appreciable poitive taining for IgG within parietal cortex (not hown), indicating that the blood brain barrier wa intact to paage of large plama protein detectable with IgG. Quantitative analyi of thee ection demontrated there wa no difference in the darkne of taining in ipilateral veru contralateral cortex for the 1 and 3 day group (.7 ± 1.2 and.1 ± 1.9 gray level, repectively) (Additional file 4).

7 Page 7 of d a b c Ipilateral H&E (Score) ** ** 3d Time pot mild d e f 6 g h i Ipilateral ED1 (Cell/Field) Ipilateral GFAP (Score) d Time pot mild 3d Time pot mild Fig. 3 Acute and ubacute cortical repone to a ingle mild ichemic inult. a, b Hematoxylin and eoin (H&E) tained ection from brain perfuion fixed at 1 or 3 day following a mild tranient middle cerebral artery occluion (). c The median and firt and third quartile of the damage core wa not different tatitically between group. d, e ED1 tained ection from brain 1 and 3 day pot. f Mean number of ED1 cell tained per field were not different tatitically. g, h GFAP tained ection from animal 1 and 3 day pot. i Median and firt and third quartile of the GFAP core were imilar between group (P >.5). n = 8/group *P <.5; **P <.5, ipilateral different from contralateral. (Mann Whitney Rank Sum tet) * Sytemic inflammation following a mild tranient ichemia Conidering the evidence that ytemic inflammation or uppreion of the immune ytem can play a role in influencing ichemic damage [21, 23, 24], we alo invetigated the potential contribution of ytemic immune cell change to the enhancement of ichemic damage by performing a hematology analyi of blood ample collected at 1 or 3 day following a mild or a ham urgery. Blood ample from a naïve group of rat provided an additional control (n = 5/group). Group had a imilar hematocrit (Fig. 5d and Additional file 5). Following a mild there wa a decreae in the lymphocyte percentage of white blood cell and the platelet concentration at 1 day but not 3 day relative to naïve control (Fig. 5a, b). In contrat, there wa a tranient increae in the granulocyte percentage of white blood cell at 1 day compared to level either in naïve animal or in blood ample collected 3 day after (Fig. 5c). Altered lymphocyte and granulocyte concentration but no change in platelet were alo oberved 1 day following the tre of a ham urgery. However, the increae in granulocyte alone wa inufficient to enhance damage; ham urgery with increaed granulocyte and a mild ichemic inult 1 day later produced only mild ichemic change (Fig. 1c). Dicuion The current reult demontrate the major importance of early recurrence of a mild tranient ichemic inult following an initial TIA. Ditinctive in our tudy, compared to tudie of preconditioning, i the invetigation of multiple tranient ichemic inult both of ufficient

8 Page 8 of 14 Contralateral Ipilateral 3 d a b c d 25 M Ipilateral TNF (Cell/Field) * * 3d Time pot mild e f 25 M i j 4 k l g 25 M m n o p 25 M h Ipilateral Iba1 (Cell/Field) Ipilateral Lectin (Score) Ipilateral EBA (Veel % Contral) * * 3d Time pot mild ** 3d Time pot mild ** ** 3d Time pot mild Fig. 4 Cerebral Inflammatory and blood brain barrier marker change following a ingle mild tranient. a Repreentative ection tained from contralateral cortex for the cytokine, tumor necroi factor (TNF). b, c TNF immunotained ection from ipilateral cortex at 1 or 3 day pot, repectively. d Mean number of TNF tained cell per field i imilarly increaed at 1 or 3 day pot (n = 8/group). Repreentative poitive taining of microglia with Iba1 in contralateral cortex e and ipilateral cortex at 1 and 3 day pot (f, g). Activated microglia were oberved at 1 day and thee were more numerou at 3 day pot (h, n = 8/group). Double taining of veel and microglia with tomato lectin (i l) and veel with endothelial barrier antigen (EBA) (m p). Staining of microglia with lectin wa minimal in the contralateral cortex (e.g. i) and increaed (e.g. arrow) in ipilateral cortex at 1 day (j) or 3 day (k) pot tranient. l Median core for lectin taining (n = 8/group). m Repreentative taining of veel for endothelial barrier antigen (EBA) in contralateral cortex (12 animal with good contralateral poitive taining quantified). n, o EBA taining in ipilateral cortex from animal at 1 and 3 day pot. p Reduced percentage of veel tained with EBA ipilaterally relative to contralaterally (n = 6/group). *P <.5; **P <.5, ipilateral different from contralateral. P <.1 lectin tained microglia differ between 1 and 3 day pot group everity to caue mild ichemic damage. We found that a recurrent mild inult following a TIA in rat produced ubtantially more brain damage when recurrence i acute (i.e. 1 day) than with a ubacute recovery time of 3 day between inult. The caue of thi difference i likely multifactorial and potentially include an augmentation of damage by ytemic inflammatory change. Irrepective of the mechanim, the reult demontrate that the time of mild troke recurrence can be crucial in influencing brain damage and upport urgency for determining and implementing optimal troke prevention management early after TIA to avoid a econd ichemic event. Interaction between two Mild Ichemic Inult The principal finding of the current tudy wa that the combined damage produced by multiple mild inult wa influenced by their timing. In contrat to that in

9 Page 9 of 14 a b Lymphocyte (%) Platelet (x1 1 /L) Naive Sham d Naive Sham d c d Granuloycte(%) Hematocrit (%) Naive Sham d Fig. 5 Sytemic change following a Single Mild Tranient. A complete blood count of blood ample from naïve animal or animal at 1 or 3 day pot a mild tranient. a The concentration of lymphocyte, preented a a % of total white blood cell, wa decreaed at 1 day pot tranient when compared to naïve animal. b The mean concentration of platelet wa alo decreaed at 1 day pot. c The mean number of granulocyte, normalized to the total number of white blood cell, wa traniently increaed at 1 day. d Mean hematocrit wa imilar in all group. N = 5/group. P <.5; P <.5, different from Naive. P <.5, different from 1 day pot (ANOVA and Bonferroni t-tet) Naive Sham d the current tudy, previou tudie of multiple ichemic inult have generally focued on tudie where the firt or econd inult i a very hort preconditioning or potconditioning ichemia that alone produce no permanent cellular damage; uch a non-damaging ichemia ha protective effect [25 28]. Indeed in the current tudy the group with a ham procedure prior to tranient had reduced damage compared to that of a ingle aeed 3 day later. Uing an initial inult that i more injuriou, one laboratory ha examined the effect of repeated ichemia in the gerbil uing only animal that diplayed neurological ign of troke after a firt temporary carotid occluion and ubjecting them to additional epiode of the ame duration of ichemia [29, 3]. Although mall number (3 5) per group were invetigated, their reult uggeted a tranition from mild to more evere injury with increaing number of inult and greater damage when econd inult were at 3 or 5 veru 48 h apart. Recently, we ued a model of two relatively hort tranient ichemic epiode, produced by microclip occluion of the MCA and eparated by 3 day to demontrate an increaed damage with recurrent troke compared to a ingle inult [15]. The preent tudy alo oberved an increae in the damage produced by a recurrent compared to a ingle, but difference were mot marked for an acute (1 day) rather than a ubacute (3 day) recurrence. Extenive infarction rather than partial infarction and/or elective necroi wa produced when troke recurrence wa acute rather than ubacute. In addition to thi finding, we oberved everal differential ytemic and cerebral inflammatory change at acute and ubacute time following a mild tranient ichemic inult providing novel inight into the pathophyiology of TIA and potential contribution to recurrent troke damage. Thee reult are of potential relevance clinically conidering an anticipated rie in the incidence of TIA, both due to our ageing population and an ecalation in number of ichemic troke patient that will be treated with early reperfuion therapy [8 1]. Undertanding better the pathophyiology of early recurrence i alo eential conidering that the median time for recurrence of functional deterioration related to a vacular event following a TIA i 1 day [14]. Furthermore, although debilitating troke are relatively infrequent following a TIA alone, when there i a recurrent event, 53 % have been found to be debilitating [31].

10 Page 1 of 14 Phyiological and vacular change Our phyiological meaure determined that variou factor known to influence infarct ize uch a body temperature, blood preure and blood gae were imilar between the group and thu are unlikely to account for the effect of different timing of recurrent inult on brain damage. Although preconditioning ichemia ha been oberved by other to improve blood flow during ichemia compared to a ingle evere ichemic inult [32, 33], the level of cerebral blood flow reduction were equivalent during both inult and for both group. With repect to reperfuion, there were no ignificant tatitical difference in perfuion within the firt 5 min of reperfuion. However, there wa a variable return of flow to baeline, with ome animal demontrating hyperperfuion and thi variance wa aociated with uboptimal tatitical power requiring caution for deciding the group mean were the ame. Alo a limitation preently wa that only acute reperfuion wa monitored and the poibility remain that at a delayed hypoperfuion could have adverely influenced damage. Regarding vacular change, there wa little evidence for difference in diruption of the blood brain barrier at early veru ubacute time. There wa a lack of detectable extravaation of IgG at either time point in agreement with previou report of a lack of vaogenic edema detectable in T 2 magnetic reonance image following a mild tranient ichemic inult [5, 7]. The reduced taining for EBA did ugget ome endothelial injury at both 1 and 3 day pot a ingle. Early ichemic endothelial injury i aociated with an acute activation of the endothelium reulting in an increaed expreion of endothelial adheion molecule aociated with endothelial injury [21, 24, 34]. However, ince reduction in EBA were imilar at both time point, there wa no direct evidence for greater dyfunction in the blood brain barrier at 1 than 3 day to help account for greater enhanced damage of a recurrent 1 day inult. Cerebral inflammatory change We alo invetigated whether the firt mild ichemic inult could produce factor in the tiue that could either augment ichemic damage acutely (i.e. at 1 day) or provide ome later (3 day) neuroprotection. The literature i pare regarding acute glial inflammatory change in the brain following a mild ichemic inult mimicking a TIA. At relatively chronic time (1 4 week) following a mild focal tranient ichemia increaed atroglial reactivity uing GFAP ha been reported previouly [5, 7, 35, 36]. Similarly, at chronic time pot-inult, microglial activation detected a poitive immunotaining for OX42 (an immune marker for CD11b which alo tain activated neutrophil and microglial derived macrophage) [37] wa increaed within area of elective necroi at 2 and 4 week pot inult followed by a decline in OX42 taining thereafter [5, 6, 36]. We invetigated acute microglial/ macrophage activation uing Iba1, tomato lectin and ED1 taining and oberved a conitent early (1 day) increae in Iba1 tained microglia but pare poitive ED-1 taining in 3/8 animal. Staining for activated microglia/macrophage uing Iba1 and lectin taining wa increaed at 3 day compared to 1 day pot a mild ichemic inult and ED-1 taining wa preent in 6/8 animal. Thi upport that microglial activation occur a early a 1 day pot a mild ichemic inult, conitent with previou obervation of early microglial activation in peri-infarct region i.e. region which likely experienced mild tranient ichemia; and, thi i followed by the appearance of macrophage within the core and peri-infarct region at 3 5 day pot-inult [37]. There i a complex and incompletely undertood progreion of activation of different microglial phenotype (e.g. macrophage that are of an M1 or M2 phenotype), however, there i agreement in general that activated microglia appear to be involved in both necrotic and repair repone [21, 37 39]. Thu it i poible that in the preent tudy, increaed microglial activation oberved at 3 day provide ome neuroprotection and act to help reduce damage during the econd inult via expreion of anti-inflammatory cytokine uch a IL-1 or TGF-β and factor uch a IGF-1 [21, 37, 39]. Future elucidation of the pathophyiology of the polarity change of the variou immune cell activated following ichemia hould conider identifying the pecific mediator within the microenvironment in repone to differing everitie of cerebral ichemia including a mild tranient ichemia. Preently, evidence wa lacking for a potential involvement of a differential releae of pro-inflammatory neurotoxic cytokine enhancing acute recurrent ichemic damage conidering taining for TNF-alpha tended to be greater rather than le at 3 day. Sytemic inflammation Examination of peripheral blood following a hort tranient ichemia indicated that even a mild ichemic inult evoked alteration in inflammatory cell within the blood. Although the complex and dynamic immune repone pot ichemia are till being characterized in detail and their role in affecting ichemic outcome remain controverial, numerou tudie indicate that ichemic alteration in peripheral inflammatory cell can modify brain damage [21, 23, 24]. Following the mild tranient ichemia produced currently, we found alteration in the concentration of lymphocyte, granulocyte and platelet in the blood. Regarding change in lymphocyte, both our ham animal and animal 1 day pot a mild ichemic inult

11 Page 11 of 14 demontrated a decreae in peripheral lymphocyte compared to our naïve control. Thi i conitent with the lymphopenia aociated with immune uppreion and an increaed uceptibility to infection that i well recognized to occur pot troke in human and experimental animal [4 43]. The reduction in lymphocyte with the current mild tranient ichemia produced by 3 min clip occluion wa rather modet (1 15 %) and hort lating compared to the >5 % reduction in lymphocyte oberved for at leat 2 week following 6 min of tranient in mice uing a tranient intraluminal thread occluion [4]. Preently, lymphocyte number returned toward naïve control level already by 3 day pot-inult. The white blood cell analyi alo provided a count of polymorphonuclear granulocyte which conit of predominantly neutrophil along with eoinophil and baophil. The increae in granulocyte oberved at 1 day pot-inult i conitent with report of increaed granulocyte following troke in human and experimental animal [41, 43 46]. The origin of the additional granulocyte can vary with the type of injury but include mobilization from the pleen and bone marrow and potentially increaed production and decreaed apoptoi [34, 37]. The mechanim mediating the increae likely include a tre repone [23, 41] which i conitent with previou report and our obervation of elevated number of granulocyte and decreaed number of lymphocyte, alo in animal ubjected to ham urgery [4, 47, 48]. With a mild tranient ichemic inult, we oberved an increae in granulocyte that wa temporary with a normalization by 3 day pot. A imilar return toward baeline wa oberved in mice with either ham urgery or uing a tranient ligature [48]. Important to note i that the tre induced neutrophilia would appear inufficient to enhance damage becaue a ham urgery 1 day prior to mild tranient reduced ichemic damage indicating that a combination of both tranient cerebral ichemia and neutrophilia would be involved in exacerbating damage. Indeed, the interaction of granulocyte or neutrophil with cerebral ichemia i a complex operation of dynamic change in neutrophil activation at variou ite (e.g. vacular and parenchymal) underlying multiple function (e.g. blood brain barrier diruption, thrombu/clot formation or neurotoxicity) [34]. Both clinical and experimental tudie have reported an increaed accumulation of neutrophil in the brain after an ichemic troke [34, 37, 49]; and, there i evidence for their role in enhancing brain damage but alo evidence for their lack of an effect or for producing beneficial effect via neuroprotection or an involvement in brain repair e.g. [34, 37, 5 53]. Thi variability may reflect the array of time dependent change in variou molecular ignal and their interaction on multiple cell type with potential difference according to pecie. Currently, the neurovacular unit i conidered a key ite of neutrophil action at delayed time following tranient cerebral ichemia. Following tranient ichemia, Ly6G poitive neutrophil infiltration into the brain parenchyma wa minimal or occurred within the vicinity of veel [54, 55]. Alo following tranient ichemia in the moue, homogenized brain ample analyzed with flow cytometry demontrated a delayed (2 3 day) increae in neutrophil not preent at 1 day [53, 56]. Note that neutrophil migration appear to occur ooner and i more intene without reperfuion; it ha been oberved early (e.g. at 1 day) following permanent ichemia [47, 57]. Although there may be a lack of migration into brain acutely following a ingle tranient ichemia, granulocyte recruitment, activation and infiltration [34, 37] on primed cerebral endothelium may be greatly accelerated by the ubequent flow diruption produced with a recurrent ichemic inult. A contribution from platelet i alo poible a upported by their decreae at 1 day compared to 3 day pot-inult poibly reflecting continued activation/interaction with cerebral ichemic vaculature. Reduced platelet count have alo been oberved in troke patient and there i evidence for their interaction with peripheral inflammatory repone and ichemic injury [58 6]. Additional reearch i required to clarify the importance of granulocyte and platelet interaction and the dynamic change in injured cerebrovacular endothelium following an initial mild ichemic inult prior to a econd TIA. The poibility for different peripheral immune repone occurring with a firt TIA veru a recurrent troke/tia hould alo be conidered [61]. Concluion To conclude, the production of multiple mild ichemic inult with different recovery time between them demontrated an important dependence on the timing between inult. The damage oberved with a hort recovery of 1 day following a mild tranient ichemia ubtantially exceeded that with a ubacute recovery time of 3 day. The reult might help explain clinical report of increaed rik of recurrent troke in the firt day following a TIA/minor troke or the udden clinical deterioration oberved in ome cae oon after troke [62, 63]. The mechanim involved in the repone are likely complex involving a progreion of ytemic and cerebral/vacular inflammatory change after the firt mild ichemic inult that affect the econd inult. Irrepective, the reult indicate that in the abence of clinically available therapeutic therapy to attenuate injury following a tranient ichemic

12 Page 12 of 14 attack or it recurrence, patient hould receive immediate care to bet manage and prevent troke recurrence e.g. with early invetigation of thromboembolic ource, intenive antiplatelet therapie, lipid lowering, and/or anticoagulation for cardioembolic troke. Additional file Additional file 1. Hitology aement for Figure 1. Shown are the data for each animal for the and 3d recurrent (Recur.) group and the Sham (h) plu mild (mld) troke group. The decriptive aement, the H&E core, the ED1 count and the GFAP core are preented. Additional file 2. Phyiological meaure for each animal in the and 3d recurrent mild ichemic inult group. Shown are the doppler flow meaure (% baeline), rectal temperature, mean arterial blood preure (MABP) and blood ga value. Additional file 3. Hitological aement for Figure 3. Shown are the data for each animal at either or 3d pot a ingle mild ichemic inult. The H&E core, the ED1 count and the GFAP core are preented. Additional file 4. Hitological aement for Figure 4. Shown are the data for each animal at either or 3d pot a ingle mild ichemic inult. Poitive taining count for TNF, Iba1 and EBA in addition to the Lectin core and IgG gray level meaure are preented. Additional file 5. Complete blood analyi value for each of the animal in each group the naïve control group, the control group with ham middle cerebral artery (MCA) urgery, the pot MCA occluion and 3d pot MCA occluion group. Preented are the number of white blood cell, lymphocyte, monocyte, granulocyte (x19/l) and the number of lymphocyte, monocyte and granuloctye (% total white blood cell). Hematocrit and number of platelet are alo preented. Abbreviation EBA: endothelial barrier antigen; GFAP: glial fibrillary acidic protein; IgG: immunoglobulin G; : middle cerebral artery occluion; TIA: tranient ichemic attack; TNF: tumor necroi factor. Author contribution All author helped to draft the manucript. In addition, UIT participated in tudy conception, deign and data interpretation and contributed to hitological aement and tatitical analyi of the data. ZZ carried out the recurrent troke urgerie and participated in coordination of the tudy and data interpretation. PAB participated in tudy conception and deign and interpretation of the tudy. MQ participated in the recurrent troke urgerie and carried out the hitological procedure and aement of hitological injury. All author read and approved the final manucript. Author detail 1 Department of Clinical Neurocience and Hotchki Brain Intitute, Cumming School of Medicine, Univerity of Calgary, Calgary, AB T2N 4N1, Canada. 2 Department of Phyiology and Pharmacology, Univerity of Calgary, Calgary, AB T2N 4N1, Canada. Acknowledgement We gratefully acknowledge the technical aitance during the urgical experiment provided by Melia Morgunov. The animal reource centre performed the hematology analyi of the blood ample. Availability of upporting data The data et upporting the reult of thi article are included within the article and it additional file. Competing interet The author declare that they have no competing interet. Ethic Thi experimental animal tudy wa carried out in accordance with the guideline and policie of the Canadian Council on Animal Care for the ethical care and handling of animal. The current tudy wa approved by the Univerity of Calgary Health Science Animal Care Committee (Protocol M1117 and AC15-43). Funding Thi tudy wa upported by funding from the Canadian Intitute for Health Reearch (Grant MOP11137). Thi funding body had no role in the deign of the tudy nor in the collection, analyi and interpretation of data nor in writing of the manucript. Received: 17 November 215 Accepted: 11 May 216 Reference 1. Black M, Wang W, Wang W. Ichemic troke: From next generation equencing and GWAS to community genomic? 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