Accepted Manuscript
Transient ischemic attack induced by melted solid lipid microparticles protects rat brains from permanent focal ischemia
Ming-Jun Tsai, Yu-Min Kuo, Yi-Hung Tsai PII: S0306-4522(14)00499-0
DOI: http://dx.doi.org/10.1016/j.neuroscience.2014.06.014X
Reference: NSC 15483 To appear in: Neuroscience Accepted Date: 6 June 2014
Please cite this article as: M-J. Tsai, Y-M. Kuo, Y-H. Tsai, Transient ischemic attack induced by melted solid lipid microparticles protects rat brains from permanent focal ischemia, Neuroscience (2014), doi:
http://dx.doi.org/ 10.1016/j.neuroscience.2014.06.014X
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TRANSIENT ISCHEMIC ATTACK INDUCED BY MELTED
SOLID LIPID MICROPARTICLES PROTECTS RAT BRAINS
FROM PERMANENT FOCAL ISCHEMIA
(Running title: transient ischemia attack protects permanent stroke)
a D ep art m en t of N eu rol og y, C hi na M ed ic al U ni ve rsi ty H os pit al, Ta ic hu ng , Ta iw an b
School of Medicine, Medical College, China Medical University, Taichung, Taiwan cDepartment of Cell Biology and Anatomy, National Cheng Kung University, Tainan,
Taiwan d
Graduate Institute of Clinical Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
Keywords: transient ischemic attack; solid lipid microparticles; ischemic
preconditioning; ischemic stroke; chitin/PLGA blended microparticles
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＊ C or re sp on de nc e: Dr . Y. -M . K uo , D ep art m en t of Ce ll Bi ol og y an d A na to m y, N ati on al C he ng Kung
University. 1 Ta Hsueh Road, Tainan, Taiwan 70101. Tel.: +886-6-2353535 ext. 5294;
fax: +886-6-2093007. E-mail address: [email protected] X
Dr. Y.-H. Tsai, Graduate Institute of Clinical Pharmacy, College of Pharmacy,
Kaohsiung Medical University, 100 Shih-chuan 1st Road, Kaohsiung, Taiwan, R.O.C.
2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
56 57 58 59 60 61 62 63 64 65 A bs tr ac t -T hi s st ud y ai m s to de ve lo p a tra ns ie nt is ch e mi c
attack (TIA) model in
conscious animals and use this model to investigate the effect of TIA on subsequent
permanent ischemia. TIA was induced by injecting designed temperature-sensitive
melted solid lipid microparticles with a melting point around body temperature into male Wistar rats via arterial cannulation. Neurologic deficit was monitored
immediately after the injection without anesthesia. According to the clinical definition
of transient ischemic attack, rats were divided into neurologic symptom durations <24 h, 24-48 h and ≥48 h groups. The lipid microparticle-induced infarct volumes were
small in the <24 h and 24-48 h groups, while the volumes were five times larger in the
≥48 h group. Permanent ischemic stroke was induced 3 d after induction of TIA by injecting a different kind of embolic particles manufactured by blending chitin and
PLGA. The <24 h group had less severe neurologic deficits and smaller infarct
volumes than that of 24-48 h and control (without prior lipid microparticle treatment) rats. Taken together, we successfully develop a TIA animal model which allows us to
TI A (< 24 h) pr ec on dit io ni ng pr ot
ects the brain from subsequent permanent ischemic stroke. 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
56 57 58 59 60 61 62 63 64 65
I
N
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D
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C
T
I
O
N
Tr an si en t is ch e mi c att ac k(TIA) is the sudden onset of transient neurologic deficit
with full recovery within 24 h (Easton et al., 2009). TIA has been recognized as a riskX
factor for permanent ischemic stroke (Easton et al., 2009). However, in the animalX model that TIA is induced by transient occlusion of ipsilateral cerebral arteries,
ischemic preconditioning has been recognized as a beneficial factor for ischemic
stroke (Perez-Pinzon et al., 1997, Kitagawa et al., 2005, Zhang et al., 2008). SeveralX clinical studies also support the premise that prodromal TIA may reduce the severity
of permanent ischemic stroke and decrease the infarct size as detected in the perfusion
scan of magnetic resonance imaging (Weih et al., 1999, Moncayo et al., 2000).X However, it has been argued that the latter studies were retrospective in nature, with most of the information describing the relationship between TIA and a following
permanent ischemic stroke being culled from medical histories or descriptions from
family members of patients. A more recent study investigating a much larger cohort failed to find the protective effects of preceded TIA on permanent ischemic stroke
ch ar ac ter iz ati on .
A major drawback of previous studies in characterizing the effect of TIA
preconditioning on permanent stroke severity is that they were unable to evaluate the
4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
56 57 58 59 60 61 62 63 64 65 ex ac t te m po ral rel ati on sh ip be tw ee n TI A an d ne ur ol og ic de fic it. In clinical studies, it
has been demonstrated that the duration of TIA-induced neurologic deficit is closely
related to the incidence of stroke. For example, TIA symptoms lasting more than one
h increase the incidence of stroke or stroke recurrence (Giles et al., 2006, Ois et al.,X
2008, Sciolla and Melis, 2008). However, few animal studies focused on the durationX
of neurologic deficit induced by TIA, because in most animal studies TIA was induced by transient occlusion of brain circulation under anesthesia. It is impossible
to evaluate the onset time of neurologic deficit secondary to TIA until hours after
recovering from anesthesia. Therefore, an animal model that permits inspection of the
behavior immediately after the induction of TIA is essential to examine the
association of preceded TIA and the severity of following ischemic stroke.
The objectives of this study are to develop a TIA animal model that allows us to evaluate the TIA-induced neurologic deficit in real-time and to evaluate the effect of
TIA on the neurologic outcomes of subsequent permanent ischemic stroke. We develop the TIA animal model by injecting artificial emboli which were designed to
m elt in vi vo aft er inj ec tio n int o
the brain circulation. Lipid microparticles, especially
solid lipid microparticles, have been used for drug delivery carriers because of their
ideal characteristics of homogenous sphere appearance and rapid and consistent
dissolvability (Jannin et al., 2008). By taking these advantages, we designed solidX
5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
56 57 58 59 60 61 62 63 64 65 lip id mi cr op art icl es th at m elt at bo dy te m pe rat ur e to pr od uc e art ifi ci al emboli. The
injection of melted solid lipid microparticles was performed without anesthesia, so the
TIA-induced neurologic deficit could be evaluated immediately from the onset of
neurologic symptoms. After completely recovered from neurologic deficit, a different type of artificial particle emboli, manufactured by chitin and
poly(D,L-Lactide-co-glycolide) (chitin/PLGA) blended microparticles, were injected
to induce permanent ischemic stroke (Tsai et al., 2011). Neurologic deficit wasX evaluated immediately after the injection of chitin/PLGA microparticles. Regional
cerebral blood flow was determined before, during, and after the injection of
microparticles. Brain infarction was assessed by triphenyl tetrazolium chloride (TTC) stain.
EXPERIMENTAL PROCEDURES
Materials
Gelucire 33/01 and Gelucire 43/01 were purchased from Gattefosse Co. (St-Priest,
fr o m Si g m a-Al dri
ch Co. (St. Louis, MO). Chitin was purchased from Tokyo Chemical
Industry Co. (Tokyo, Japan). Tetrazolium Red (2,3,5-triphenyl-tetrazolium Chloride)
was obtained from Alfa Aesar Co. (Ward Hill, MA). All other chemicals and solvents
6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
56 57 58 59 60 61 62 63 64 65 w er e of an al yti ca l gr ad e an d pu rc ha se d fr o m Si g m a-Al dri ch Co.
Preparation of melted solid lipid microparticles for TIA
The solid lipid microparticles were made by adding the oil phase into the water phase
containing hydrophilic surfactant. The preparations were manufactured with the rationale that the used lipids and surfactant would be biocompatible and
biodegradable. Gelucires have been used as surfactants, co-surfactants and lipid
matrixes in drug delivery systems and are generally recognized as safe (Shimpi et al.,X
2005). The oil phase was prepared by mixing Gelucire 43/01 (melting point ofX 43°C/hydrophile-lipophile balances of 1) and Gelucire 33/01 (melting point of 33°C/hydrophile-lipophile balances of 1). Gelucires are solid lipid materials
composed of mono-, di-, and triglycerides and mono- and di-fatty acid esters of
polyethylene glycol (Jannin et al., 2008). A ratio of 1:1 between Gelucire 43/01 andX Gelucire 33/01 was used to produce solid lipid microparticles that have a melting
La bo rat or y D ev ic es, In c., Ta rz
ana, CA). The blended oil phase was homogenously
mixed by heating the Glucire lipids above their melting point. The water phase was
prepared by adding sodium dodecyl sulfate in purified water to get a final
concentration of 1%. Both the oil phase and water phase were kept at 70°C and were
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56 57 58 59 60 61 62 63 64 65 mi xe d by sl o wl y dr ip pi ng th e oil ph as e di re ctl y int o th e w at er
phase while stirring at
the speed of 300 rpm for 5 min. The temperature was then allowed to slowly cool
down to room temperature. The gelled lipid microparticles were allowed to harden in
the cool water phase while stirring at the same speed for another 24 h. After hardening, the microparticles were filtered and rinsed with deionized water and the particles were
sieved by standard U.S. size meshes from 20 to 355 mesh (Analytical Test, Retsch,
Germany) before drying overnight. The finished microparticles were kept at 4°C in the refrigerator. The particle size of 75-90 μm was used as emboli for inducing
transient brain ischemia.
Differential scanning calorimetry of melted solid lipid microparticles
The thermal transitions of the lipid microparticles were analyzed by differential
scanning calorimetry. The melted solid lipid nanoparticles were freeze-dried before the measurement. The differential scanning calorimetry was performed by a
w er e pu t in al u mi nu m pa ns
and the thermal profiles were obtained as the temperature increasing from 10 to 55°C at a rate of 5°C/min under nitrogen.
Scanning electronic microscopy of melted solid lipid microparticles
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42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 T hi
s method has been described previously (Tsai et al., 2012). The melted solid lipidX microparticles were attached onto double-sided adhesive tape and fixed to an aluminum stage. The microparticles were sputter-coated with gold using a Hitachi
coating unit and the surface of the microparticles were examined using a Hitachi
S-2300 Scanning electronic microscopy (Japan).
Preparation of chitin/PLGA microparticles for ischemic stroke
The detailed procedure to prepare chitin/PLGA microparticles has been described
elsewhere (Tsai et al., 2011). Briefly, chitin powder was dissolved inX dimethylacetamide solution containing 5% LiCl to make 1% chitin solution.
Chitin/PLGA 50/50 mixed solution was formulated by directly suspending the PLGA
50/50 powder in the prepared chitin solution in 1:1 ratio. To produce microparticles, chitin/PLGA 50/50 mixed solution maintained at 70°C was dropped through a syringe
(27 gauge) into 1% sodium lauryl sulfate solution bath maintained at 25°C, and then
si ev ed by st an da rd U. S. si ze m
eshes from 40 to 400 mesh (Analytical Test, Retsch, Germany). Animal model 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Al l
experimental protocols were performed according to National Institutes of Health Guideline for animal research (Guide for the Care and Use of Laboratory Animals)
and approved by the Kaohsiung Medical University Institutional Animal Care and
Use Committee. Male Wister rats (3-month-old, 300-350g) were used in this study. All animals were housed in a temperature-controlled animal care facility under
diurnal lighting and given free access to food and water.
The surgical procedures have been described previously (Tsai et al., 2011). InX short, all animals were made to fast overnight and were anesthetized by an
intra-peritoneal injection of 300 mg/kg choral hydrate. During operation, the body
temperature was maintained at 37°C by an automated temperature regulation system. The hair of the rat was cut and head skin was excised from the midline to expose the
skull. Povidone-iodine (10%) was used for local sterilization. The skull was thinned
and two miniature probe-holders of the Laser Doppler instrument (MBF3D, Moor Instruments, Ltd., Axminster, UK) were fixed on the skull, 3 mm lateral and 0.5 mm
mi ni ho ld er ex po se d. Te tra cy cli
n HCL (1%) ointment was applied to the wound 2-3 times per day to prevent infection.
The rat was then fixed in supine position on an operation plate and midline
excision of ventral neck was made to expose the bifurcation of right carotid artery.
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42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 T he P
E-10 catheter (Becton Dickinson, NJ, USA) was inserted through the external
carotid artery (ECA) to the bifurcation of the common carotid artery (CCA), while the
four ipsilateral collateral cerebral arteries were ligated. The PE-10 catheter was then
secured by one end fixed to the ECA and the other end exposed on the neck as an indwelling injection tube after the closure of the surgical wound. Fifty microliter of
heparin solution (50 I.U./ml) was administered in order to prevent clotting on the
PE-10 tube. Surgical wounds were carefully cleaned to prevent infection. After recovery from the operation, all rats received a neurologic evaluation. If
any neurologic deficits were noticed, the rats would be excluded from further studies.
We induced TIA and ischemic stroke without anesthesia by injecting artificial emboli through the secured PE10 tube.
Laser Doppler instrument for monitoring cerebral blood flow
Local cerebral blood flow in the middle cerebral artery was initially monitored before
ce re br al bl oo d fl o w w as re
corded immediately before the injection of microparticles
and then again after injection at the following time points: 5, 10, 15, 20, 25 and 30
min and 1, 2, 4, 24 and 48 h. Throughout the entire monitoring process, the rat was
awake without anesthesia.
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42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Es ti m
ation of the duration of ischemia-induced neurologic deficit
For inducing transient brain ischemia, we injected 0.5 mg melted solid lipid
microparticles (75-90 μm in diameter) via the indwelling PE10 tube without
anesthesia. Neurologic deficit was evaluated immediately after the injection (0 min) and at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 min and 2, 3, 4, 5, 6, 7, 8, 12,
24, 36 and 48 h post injection. We graded the neurologic deficit of the rats using the
following criteria: 0 – no neurologic defect, 1 – one paw clumsiness or clasping during tail suspension, 2 – tilt, 3 – rounding in only a unilateral circle, 4 – akinesia,
5 – seizure, 6 – stupor or lack of any spontaneous movement, 7 – death. The rats that
died within 3 h after surgery were withdrawn from the study. All rats were evaluated randomly by the same investigator.
Permanent ischemic stroke was induced 3 days later after full recovery from
transient ischemia induced neurologic deficit. We injected 0.5 mg Chitin/PLGA blend
particles (75-90 μm in diameter) via the indwelling PE 10 tube to induce ischemic
ne ur ol og ic de fic it fr o m isc he
mic stroke was the same as transient brain ischemia.
Calculation of the infarction volume of the rat brain
The procedure for calculation of infarction volume has been described elsewhere
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42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 (T
sai et al., 2011). In short, the rat brains were removed and positioned in a rat brainX matrix (Activational Systems, Ann Arbor, MI) after deep anesthesia. Each brain was cut, from the tip of olfactory bulb to the caudal end of cerebellum, into 12 coronal
sections, 2 mm thick. The sections were stained by 0.05% TTC solution for 30 min
and fixed in 4% formaldehyde solution for 24 h. The infarct areas were traced and quantified by an image analysis system (ImageJ v. 1.36b version, NIH, USA). For
interpreting the infarction volume of different functional areas, the infarct area of each
functional area including the cortex, basal ganglia & thalamus, hippocampus, and cerebellum & brain stem were calculated separately. We obtained final infarct volume
after correcting with cerebral edema.
Statistical analyses
All data were expressed as mean ± SEM. The behavior score of the four different
symptom duration groups or the four pretreatment groups were assayed using
fa ct or an d gr ou p as be tw ee n su
bject factor. Bonferroni posttest was used to compare
between subject factors at each time point. The results of infarct volume in four
different groups were analyzed by two-way ANOVA followed by Bonferroni posttest.
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41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Results
Physicochemical characteristics of the melted solid lipid microparticles
The solid lipid microparticles were produced by emulsifying the oil phase, prepared
from a mixture of different melting points of Gelucire, and the water phase,
containing hydrophilic surfactant. We mixed Gelucire 33/01 and Gelucire 43/01 at a
ratio of 1:1 to form solid lipid microparticles with a melting point of 38°C and
hydrophile-lipophile balances of 1 (38/01). Differential scanning calorimetry revealed that the melting process took place with a maximum peak at 34.8°C for Gelucire
43/01 and 20.8°C for Gelucire 33/01 with a shoulder peak at 24.5°C (Fig. 1A). The
thermograms of blended Gelucires (38/01) revealed a large endothermic event from 13.8°C to 42.3°C with two distinct polymorphs melting at different temperatures.
Gradation sieving of the solid lipid microparticles yielded 15 size fractions
ranging from 20 μm to > 355 μm (Fig. 1B). The distribution of the amount of each size fraction was skewed; more than half of the particles produced by our method
mi cr op art icl es ha d un ifo rm ro un
d shapes with porous surface (Fig. 1C).
An animal model of TIA induced by melted lipid microparticle embolization
Thirty-one rats received a PE-10 tube catheterization in the neck and two miniature
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42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 pr ob
e-holders on the thinned skull windows. Three rats were excluded from the study due to failure of PE-10 catheter insertion during anesthesia or catheter misplacement.
To induce TIA, 0.5 mg of blended Gelucire (38/01) melted solid lipid microparticles
within the range of 75-90 μm (lipid particles75-90μm) were delivered to the PE-10 tube without anesthesia. We chose the Gelucire (38/01) melted solid lipid particles75-90μm
because of their ability to melt quickly at body temperature. We used the particle size
of 75-90 μm to induce embolic ischemia because previously we have demonstrated that particles at these size ranges induced an unique pattern of infarction resembling
lacunar infarcts (Tsai et al., 2011). Neurologic deficit was evaluated immediately afterX the injection of lipid particles75-90μm until the ischemia-associated behavior was no
longer evident. All 28 rats showed signs of neurologic deficit with different durations.
Based on the symptom duration (the latest time to observe any neurologic deficit), the
28 rats were divided into 3 groups (Fig. 2A): 1) 12 rats fully recovered from
neurologic deficit within 24 h (<24 h), 2) 10 rats fully recovered between 24-48 h (24-48 h) and 3) 6 rats still showed signs of neurologic deficit at 48 h (≥48 h). We did
La se r D op pl er
flowmetry showed that the blood flows of both hemispheres were
unchanged after the inserting of PE-10 tube (Figure 3). Injection of lipid particles75-90 μm reduced cerebral blood flow in the ipsilateral hemisphere with different patterns
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47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 (F ig ur e 3 A) . M ix ed m
odel ANOVA revealed that there were significant effects of
duration group (F = 43.3, d.f. 2/84, p < 0.001) and post-injection time (F = 3.8, d.f.
11/84, p < 0.001) on the blood flow with no significant interaction between these two
factors (F = 1.3, d.f. 22/84, p = 0.473). Bonferroni’s post-hoc tests showed significant changes in cerebral blood flow between <24 h and ≥48 h groups (Figure 3B). No
difference between 24-48 h and <24 h groups or 24-48 h and ≥48 h groups was found.
On the average, the blood flow of the <24 h group completely restored in about 1 h and the 24-48 h group in about 4 h after the injection of lipid particles75-90μm,
indicating the melting of microparticles successfully ensued after embolization. Reduction of cerebral blood flow was still noticeable 48 h after the injection in the
≥48 h group (Figure 3B).
Neurologic deficits in rats with different durations of brain ischemia
The average neurologic scores of the three groups were shown in Figure 4A. Except
some rats in the <24 h group, all rats showed some signs of neurologic deficit within 1
th er e w er e si gn ifi
cant effects of duration group (F = 269.6, d.f. 2/700, p < 0.001) and
post-injection time (F = 10.3, d.f. 27/700, p < 0.001) on the behavior score (Fig. 4A).
A significant interaction between these two factors (F = 1.8, d.f. 54/700, p < 0.001)
16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 w as ev id en t, su gg
esting that symptom duration groups have different effects on
behavior score. Bonferroni’s post-hoc tests showed that the <24 h group had lower
behavior scores (less severe) than that of 24-48 h and ≥48 h groups, whereas, the
24-48 h and ≥48 h groups had comparable behavior scores except at the 36 and 48 h post-injection time points (Fig. 4A).
Lipid particles75-90μm injection induced lacuna-like small infarctions primarily in
the cortex and basal ganglia & thalamus regions; occasionally, infarctions were observed in the hippocampus and cerebellum & brain stem regions (Fig. 4B).
Two-way ANOVA revealed that there were significant effects of duration group (F =
27.2, d.f. 2/125, p < 0.001) and brain region (F = 12.5, d.f. 4/125, p < 0.001) on the infarct volume (Fig. 4C). A significant interaction between these two factors (F =
5.1, d.f. 8/125, p < 0.001) was evident, suggesting that symptom duration groups have different effects on infarct volume. Bonferroni’s post-hoc test showed that the >48 h
group had larger infarct volume in the whole brain and cortex than that of the <24 h
and 24-48 h groups (Fig. 4C). No difference in infarct volume could be found in any brain region between the <24 h and the 24-48 h groups.
Di
fferent durations of preceded brain ischemia induce differential protection
against permanent ischemic stroke
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49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 W e th en in ve sti ga te th e eff ec t of
preceded brain ischemia on the severity of
neurologic deficit of the following permanent ischemic stroke in a different batch of
59 rats. Among them, nine rats were excluded from the study because of PE-10
catheter insertion failure during anesthesia, catheter drop or death due to anesthesia. The remaining 50 rats were divided into two groups: 12 rats served as saline controls,
while the other 38 rats were subjected to TIA induction. Immediately after the lipid particles75-90μm injection, all 38 rats displayed neurologic signs: 21 rats fully
recovered from neurologic deficit within 24 h (<24 h), 9 rats fully recovered between
24-48 h (24-48 h) and 8 rats still showed signs of neurologic deficit at 48 h (≥48 h). We excluded the ≥48 h group from the following studies because these animals
showed an extended duration of neurologic symptoms and large infarct volume that
would interfere with the evaluations of the following ischemic stroke. Permanent ischemic stroke was induced in all 42 rats (control group 12 rats, <24 h group 21 rats and 24-48 h group 9 rats), 72 h after the injection of lipid particles75-90μm, by
chitin/PLGA microparticle75-90μm embolization as described previously (Tsai et al.,X
La se r D op pl er flo w
metry showed that saline injection did not alter the cerebral
blood flow (Figure 5A), while the changing patterns of cerebral blood flow of the <24
h and 24-48 h groups were similar to those shown in Figure 3B. At 72 h after the first
18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 inj ec tio n of lip id pa
rticles75-90 μm, a second injection of the chitin/PLGA
microparticle75-90 μm was given to the rats. Within 5 min after the second injection,
the cerebral blood flow levels reduced to less than 50% in all three groups and lasted
for more than 4 h (Figure 5B). Cerebral blood flow levels in the <24 h group were completely recovered and significantly higher than that of the saline injected control
group at 24 h after the injection of the chitin/PLGA microparticle75-90 μm (Figure
5B).
Almost immediately after the injection of chitin/PLGA microparticle75-90μm, rats
exhibited signs of neurologic deficits. The severity of the neurologic symptom of the
control group gradually increases in the first 4 h after the injection of the chitin/PLGA microparticle75-90μm and remained constant up to 24 h (Fig. 6A, Control). Both
symptom duration (F = 288.9, d.f. 2/1014, p < 0.001) and post-injection time (F = 12.7, d.f. 25/1014, p < 0.001) had significant effects on the behavior score, with a
significant interaction (F = 2.7, d.f. 50/1014, p < 0.001) between these two factors.
However, this interacting effect was mainly due to the increased neurologic symptoms
tra ns ie nt is ch e mi a
groups over time (F = 0.4, d.f. 24/700, p > 0.5) (Fig. 6A).
Bonferroni’s post-hoc test showed that the <24 h group had less sever neurologic
symptoms than that of the control group since 20 min post-injection time; while no
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45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 su ch pr ot ec tio n w
as noticed in the 24-48 h group (Fig. 6A).
The TTC-negative infarction regions, induced by the chitin/PLGA
microparticle75-90μm, spread over the four brain areas primarily in the cortex (Fig. 6B,
Control). Both preceded brain ischemia duration (F = 20.7, d.f. 2/195, p < 0.001) and brain region (F = 32.8, d.f. 4/195, p < 0.001) had significant effects on the infarct
volume (Fig. 6C). Bonferroni’s post-hoc test showed that the <24 h group had less
total and cortical infarct areas than that of controls; whereas, the 24-48 h and control groups had similar infarct volumes in all investigated brain regions (Fig. 6C).
The American Heart Association/American Stroke Association Stroke Council
defines TIA as a transient episode of neurological dysfunction caused by focal brain
ischemia without acute infarction (Easton et al., 2009). In this study, we used the “aX priori” experimental design and classified the rats with symptom duration <24 h as
TIA without knowing the infarct volume. As clinically TIA is indistinguishable from cerebral infarction with transient signs (CITS), which is characterized by symptoms
and signs resolving within 24 h despite residual brain infarction, the <24 h group may
th er e w as a gr ou
p of rats that fully recovered from neurologic deficit in less than 5 h
(hereafter designated as <5 h group). The <5 h rats restored their blood flow in less
than 30 min and no infarct could be detected in any of the four brain areas.
20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fu rth er m or e, in th
e chitin/PLGA microparticle-induced permanent ischemic stroke,
the <5 h group had less sever neurologic symptoms than that of the rest of the <24 h
(hereafter designated as 5-24 h) rats (Fig. 6D). Marginal differences (p = 0.07) in the
chitin/PLGA microparticle-induced infarct areas were also evident between the <5 h and 5-24 h groups. By comparing the neurologic symptoms of the <5 h, 5-24 and
24-48 groups, we found that the severity of neurologic symptoms increased by
increasing the TIA duration (Fig. 6D). These results suggest, within the 24 h critical window, the shorter the TIA duration, the stronger the protection against subsequent
ischemic stroke.
DISCUSSION
It has been suggested that TIA may improve the outcomes of following ischemic stroke in animals. However, the benefits of TIA preconditioning are still unclear in
clinical studies. One possible reason for such discrepancy is the unknown association between the duration of TIA and the outcome of permanent ischemic stroke -- most
an im al an d cli ni ca l st ud ies w
ere unable to get exact duration of neurologic deficit
induced by TIA. To target this question, we designed an animal model that allowed us
to monitor the neurologic behavior in real-time after the injection of emboli. Most of
the previous animal models could only evaluate permanent disability as a research
21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 tar ge t,
because animals take hours to completely recovery from the surgery. In contrast, our model evaluates the functional recovery in neurologic deficiency
immediately after transient ischemia occurred in the brain. Furthermore, our model
allows us to track a process of functional recovery in a very mild brain injury secondary to ischemia that may gain complete recovery shortly after a stroke occurs.
These mildly injured animals are often ignored or go undetected in previous models because these animals may not demonstrate permanent disability or infarction
assessed by TTC stain. Therefore, our paradigm is an original and novel model
because it provides the opportunity to evaluate functional recovery in real-time secondary to transient ischemia.
We developed thermo-sensitive solid lipid microparticles that are ready-to-melt
in body temperature as emboli to induce transient brain ischemia. The chemical and physical characteristics of the lipid microparticles imitate the clinical emboli. In
clinical situations, microembolism is recognized as an important cause of TIA (deX
Bruijn et al., 2006), and lipid-rich emboli derived from the heart with atrial fibrillationX
cr ea te d co nd iti on
s very similar to the clinical definition of TIA. The lipid
microparticle-induced reduction of blood flow restored within a few hours in most
treated animals (78.5% of rats in <24 h and 24-48 h groups), indicating the melting of
22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 mi cr op art icl es su cc
essfully ensued after embolization. These animals developed
symptoms of transient brain ischemia while the rats were not under the effects of
anesthesia. In contrast to most animal studies that only estimate the duration of
occlusion of cerebral circulation reflecting the state of impairment of blood supply to the focal brain region, we estimate the duration of neurological symptoms reflecting
the state of neurologic function recovery. Compared with the occlusion time, the
duration of neurological deficits resembles better to the real situation of clinical TIA. Furthermore, both the <24 h and 24-48 h groups had only a small volume of infarct
area (< 20mm3), very similar to the pathologic findings in the clinical TIA/CITS patients, in which up to 50% of TIA/CITS patients having abnormal brain image
findings only showed minor degree of cerebral infarction (Easton et al., 2009). TakenX
together, our animal model of transient brain ischemia not only mimics the real
situation of TIA in humans, but also resembles well to the pathogenesis of TIA. The primary objective of this study was to develop a TIA animal model allowing
us to monitor neurologic deficit in real-time. We then used this model to validate the
pr ec on dit io ni ng se
ems unlikely to be adopted in clinical application, this reproducible
model offers a platform upon which we can study the mechanisms that underlie the
protective effect of TIA preconditioning. Previous studies have suggested that repair
23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 m ec ha ni s m s of
the body may play key roles in the effect of preconditioning, similar to
the “hormesis” effect observed in toxicology (Mattson, 2008, HYPERLINK \l "page30" Ristow et al., 2009).X
Both preconditioning and hormesis suggest that stressor/toxin when given at a level
below its harmful/toxic threshold may activate or upregulate defense mechanisms. The harmful threshold varies from stressor to stressor and depends on the body’s
defense capacity. Importantly, upregulated defense mechanisms not only fix the
stressor-induced injuries, but also protect damages subsequently caused by other
similar stressors (Mattson, 2008). For example, exercise induces energy crisis,X hypoxia, hyperthermia, dehydration and oxidative stress; all are potentially harmful to
the body. However, regular exercise at moderate intensity increases the body’s defense capacity at multiple levels, such as anti-oxidation, energy reserves, trophic
factors, etc. Thus, regular exercise can also be considered as a kind of preconditioning
(Mattson, 2008, Ristow et al., 2009). Any reagent or treatment that induces similarX biological responses as in TIA preconditioning could potentially benefit the brain, especially in those who are at risk of ischemic attack. The idea of hypoxia
pr ec on dit io ni ng ha s be en e
mployed to enhance cardiac function in humans. When
humans were subjected to hypoxic exercise training, the functions of cardiac/muscular
hemodynamic adaptations and circulating progenitor cells were enhanced (Wang et al.,X
2014). The levels of angiogenic factors were also increased by hypoxic exerciseX
24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 tra ini
ng (Wang et al., 2014). The intermittent hypoxic intervention regimen mayX
induce similar responses as TIA and become a beneficial means to lessen the severity
of ischemia-induced injuries in brain.
It is interesting to note that although the rats of 24-48 h group had more severe
neurologic symptoms than that of the <24 h group, the total infarct volumes of these two groups were comparable. Detailed analysis revealed a differential distribution of
infarct area in these two groups. In the <24 h group, more than 90% of infarct area
located in the cortex; whereas, in the 24-48 h group, only about 60% of infarct area was in the cortex (Fig. 3B,C). The other major (~35%) infarct area was in the basal
ganglia & thalamus. Similar distribution pattern (involving the subcortical area) was
also noticed in the ≥48 h group whose cortical area also occupied ~60% of total infarct area. As basal ganglia & thalamus are involved in motor execution, it is not
surprising to see that infarction occurring in these two areas gives a more severe
motor-related neurologic symptom than that occurring in the cortical area. Whether the differential distribution of infarct area determines the precondition-associated
pr ot ec tio n, as ob se rv ed in fig ur e
4, awaits further investigation. Nonetheless, our
results indicate that the duration of TIA is an important determine factor to predictor
the outcomes of subsequent permanent ischemic stroke.
25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 CONCLUSION
We have developed an animal model that allows us to monitor the neurologic
symptoms immediately after the injection of emboli. This animal model also allows
us to evaluate the influence of the neurologic symptom duration on the outcome of the following ischemic stroke. Furthermore, our model allows us to track a process of
functional recovery in a very mild brain injury that may gain complete recovery
shortly after a stroke occurs. When neurologic deficit of transient ischemia is recovered within 24 h, transient occlusion of cerebral circulation prior to ischemic
stroke is beneficial to the outcome of ischemic stroke. If the neurologic symptom of transient ischemia lasts longer than 24 h, there is no protection against following
ischemic stroke. Furthermore, within the 24 h window, the duration of preceded
transient ischemia-induced neurological deficits is reversely related to the outcome of following ischemic stroke. This animal model would provide a better platform to
A ck no wl ed g m en ts
This work was supported by National Science Council (98-2320-B-006-017-MY3 and NSC 98-2320-B-037-015) of Taiwan, Republic of China. We thank Ms. Tiffany Hu
for reading and commenting on the manuscript.
26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 R E F ERENCES
de Bruijn SF, Agema WR, Lammers GJ, van der Wall EE, Wolterbeek R, Holman ER,
Bollen EL, Bax JJ (2006) Transesophageal echocardiography is superior to
transthoracic echocardiography in management of patients of any age with transient ischemic attack or stroke. Stroke 37:2531-2534.
Easton JD, Saver JL, Albers GW, Alberts MJ, Chaturvedi S, Feldmann E, Hatsukami
TS, Higashida RT, Johnston SC, Kidwell CS, Lutsep HL, Miller E, Sacco RL (2009) Definition and Evaluation of Transient Ischemic Attack: A Scientific
Statement for Healthcare Professionals From the American Heart
Association/American Stroke Association Stroke Council; Council on
Cardiovascular Surgery and Anesthesia; Council on Cardiovascular Radiology
and Intervention; Council on Cardiovascular Nursing; and the Interdisciplinary
Council on Peripheral Vascular Disease: The American Academy of
Neurology affirms the value of this statement as an educational tool for neurologists. Stroke 40:2276-2293.
Gi le s M F, Fl os s m an
E, Rothwell PM (2006) Patient behavior immediately after
transient ischemic attack according to clinical characteristics, perception of the
event, and predicted risk of stroke. Stroke 37:1254-1260.
27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Ja nn in V, M us ak ha
nian J, Marchaud D (2008) Approaches for the development of
solid and semi-solid lipid-based formulations. Advanced drug delivery reviews
60:734-746.
Johnston SC (2004) Ischemic preconditioning from transient ischemic attacks? Data from the Northern California TIA Study. Stroke 35:2680-2682.
Kitagawa K, Yagita Y, Sasaki T, Sugiura S, Omura-Matsuoka E, Mabuchi T,
Matsushita K, Hori M (2005) Chronic mild reduction of cerebral perfusion pressure induces ischemic tolerance in focal cerebral ischemia. Stroke
36:2270-2274.
Mattson MP (2008) Hormesis defined. Ageing research reviews 7:1-7.
Moncayo J, de Freitas GR, Bogousslavsky J, Altieri M, van Melle G (2000) Do
transient ischemic attacks have a neuroprotective effect? Neurology
54:2089-2094.
Ois A, Gomis M, Rodriguez-Campello A, Cuadrado-Godia E, Jimenez-Conde J,
ris k of re cu rr en ce in pa tie nt s
with transient ischemic attack or minor stroke. Stroke 39:1717-1721.
Perez-Pinzon MA, Xu GP, Dietrich WD, Rosenthal M, Sick TJ (1997) Rapid
preconditioning protects rats against ischemic neuronal damage after 3 but not
28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 da ys
of reperfusion following global cerebral ischemia. J Cereb Blood Flow Metab 17:175-182.
Ristow M, Zarse K, Oberbach A, Kloting N, Birringer M, Kiehntopf M, Stumvoll M,
Kahn CR, Bluher M (2009) Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl Acad Sci U S A 106:8665-8670.
Sciolla R, Melis F (2008) Rapid identification of high-risk transient ischemic attacks:
prospective validation of the ABCD score. Stroke 39:297-302.
Shimpi SL, Chauhan B, Mahadik KR, Paradkar A (2005) Stabilization and improved
in vivo performance of amorphous etoricoxib using Gelucire 50/13.
Pharmaceutical research 22:1727-1734.
Tsai MJ, Tsai YH, Kuo YM (2011) Characterization of the pattern of ischemic stroke
induced by artificial particle embolization in the rat brain. Biomaterials
32:6381-6388.
Tsai MJ, Wu PC, Huang YB, Chang JS, Lin CL, Tsai YH, Fang JY (2012) Baicalein
sta bil ity an d br ai n tar ge tin g. Int J Pharm 423:461-470.
Wang J-S, Lee M-Y, Lien H-Y, Weng T-P (2014) Hypoxic exercise training improves
cardiac/muscular hemodynamics and is associated with modulated circulating
progenitor cells in sedentary men. Int J Cardiol 170:315-323.
29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 W eih M,
Kallenberg K, Bergk A, Dirnagl U, Harms L, Wernecke KD, Einhaupl KM
(1999) Attenuated stroke severity after prodromal TIA: a role for ischemic
tolerance in the brain? Stroke 30:1851-1854.
Zhang J, Yang ZJ, Klaus JA, Koehler RC, Huang J (2008) Delayed tolerance with repetitive transient focal ischemic preconditioning in the mouse. Stroke
30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fi gu re le ge n ds
Figure 1. Physicochemical and morphologic characters of Gelucire lipid
microparticles.A) The thermal transitions of Gelucire lipid excipients determined
by differential scanning calorimeter. B) Distribution of sizes of melted solid lipid microparticles (Gelucire 38/01) produced by mixing Gelucire 43/01 and Gelucire
33/01. Particles size was classified by different sizes of mesh. The distribution tends to have a skewed type (n = 3). C) Representative scanning electric micrograph of
31 rec eiv ed lip id pa rti cle s75 -90 μm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Figure 2. Neurologic deficit durations after the injection of melted solid lipid
microparticles (75-90 μm, Gelucire 38/01) into the cerebral circulation. A)
Grouping of rats. According to the definition of TIA, rats that received lipid
particles75-90μm injection were assigned, based on their symptom duration (the latest
neurologic deficit observing time), into three groups: 1) <24 h, neurologic deficit fully
recovered within 24 h; 2) 24-48 h, neurologic deficit fully recovered between 24-48 h;
3) ≥48 h, neurologic deficit was still apparent at 48 h. Each dot or circle represents an animal.B) Temporal profiles of body temperature of the three groups of rats that
32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fi gu re 3. Te m po
ral profiles of blood flow changes after the injection of melted
solid lipid microparticles (75-90 μm, Gelucire 38/01) into the cerebral circulation.
Representative temporal profiles of cerebral blood flow, determined by laser Doppler
flowmeter, in both ipsilateral (ipsi-) and contralateral (contra-) hemispheres of the three groups of rats (<24 h, 24-48 h and ≥48 h). Lower right panel shows the changes
of cerebral blood flow in the ipsilateral hemispheres of rats of three groups. ***, p <
33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fi gu re 4. N eu rol
ogic symptoms and infarct volume of rats with transient ischemic
attack induced by melted solid lipid microparticles (75-90 μm, Gelucire 38/01)
embolization. A) Temporal profiles of the severity of neurologic symptoms of the
three groups of animals with different symptom durations (Fig. 2A). B)
Representative TTC-stained serial brain sections (sections 3-7 of total 12 sections in
one rat, 2 mm thickness) display lacunar-like infarction areas (arrows) of the three
different symptom duration groups. Arrows mark the lacunar-like infarction areas.
C) Infarct volumes in four main brain regions induced by melted solid lipid
microparticles75-90 μm emboli. Four main brain regions include cortex, basal ganglia
& thalamus (BG & Tha), hippocampus (Hippo), and cerebellum & brain stem (Cb & BS). ***, p < 0.001, Bonferroni’s post-hoc tests, vs. the respective brain regions of
34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fi gu re 5. Te m po ral pr ofi les of
blood flow changes after the injections of
microparticles into the cerebral circulation. A) The changes of cerebral blood flow
in the ipsilateral hemispheres of rats that received saline injection (Control) or melted
solid lipid microparticle75-90 μm injections (<24 h and 24-48 h, see Fig. 2A for
definition). B) The changes of cerebral blood flow in the ipsilateral hemispheres of rats that received chitin/PLGA microparticle75-90 μm, 72 h after the injection of lipid microparticle75-90 μm. ***, p < 0.001, Bonferroni’s post-hoc tests.
35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Fi gu re 6. Ef fe cts of va
rious durations of prior transient ischemia on the
neurological deficits of rats with following ischemic stroke induced by chitin/PLGA
microparticle75-90μm embolization. A) Temporal profiles of ischemic stroke-induced
neurologic symptoms of the animals with different durations of prior TIA. **, p < 0.01, Bonferroni’s post-hoc test, vs. the respective time point of the control group.
B) Representative TTC-stained serial brain sections (2 mm thickness) display
ischemic stroke-induced infarction areas of the animals with different durations of prior TIA. C) Infarct volumes of animals induced by ischemic stroke with different
durations of prior TIA. Four main brain regions include cortex, basal ganglia &
thalamus (BG & Tha), hippocampus (Hippo), and cerebellum & brain stem (Cb & BS). ***, p < 0.001, Bonferroni’s post-hoc tests, vs. the respective brain regions of
the control and 24-48 h groups. D) Temporal profiles of ischemic stroke-induced neurologic symptoms of the animals with different durations of prior TIA. Mixed
model ANOVA revealed significant differences among the duration group (F = 335.5,
Highlights
Injection of temperature-melted lipid particles induce transient ischemic attack Neurologic deficit is monitored in conscious rats
Injection of blending chitin/PLGA particles induce permanent ischemia Transient ischemia (<24 h) preconditioning protects following ischemic attack
