I N A T R A N S I E N T F O R E B R A I N I S C H E M I A - R E P E R F U S I O N M O D E L I N T H E R A T
N azire Afşar, M.D.* / Sevinç Aktan, M.D.* / Dilek İnce-Günal, M.D.* Çiğdem Çelikel, M.D.** / Tam er Coşkun, M.D.*** / Berrak Yeğen, M.D.***
* D e p a r t m e n t o f n e u r o l o g y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y .
** D e p a r t m e n t o f P a t h o l o g y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y . * * * D e p a r t m e n t o f P h y s i o l o g y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y .
ABSTRACT
Objective: Cerebral ischemia results in both
necrotic and apoptotic cell death. It has been suggested that approaches directed at disrupting the apoptotic process and expression of proapoptotic proteins might be beneficial for preserving functional neuronal tissue after an ischemic insult. The aim was to evaluate the presence of apoptotic cell death and the pattern of expression of proapoptotic protein (bax) in a transient forebrain cerebral ischemia model and to observe the potential benefits of a neurotrophic factor IGF-I on these parameters.
Methods: Female/male Wistar rats weighing
200-240g were subjected to transient forebrain ischemia by bilateral carotid artery occlusion combined with systemic hypotension for 10 minutes. Three reperfusion periods were performed as 1h, 24h and 7 days. The experiment was then conducted in two arms: in group I (n=6 for each reperfusion group), intracisternal injection of vehicle or 10 pg/rat of IGF-I was performed at all reperfusion periods, and these rats were evaluated for the presence
of apoptosis and bax protein expression. Group II (n=4 for each reperfusion group) was evaluated for protein oxidation at the three reperfusion periods.
Results: Apoptosis was significantly higher
(p<0.01) in the vehicle group compared to the sham group, and IGF-I treatment resulted in a significant decrease of apoptosis compared to the vehicle treated group at 24 hour reperfusion. Moreover, a peak in apoptotic cell death at 24 hour reperfusion was observed, however remaining just short of significance (p = 0.0730). No difference in bax protein expression and protein oxidation could be demonstrated between reperfusion periods and after IGF-I use.
Conclusion: 10pg/rat of IGF-I produces a
significant suppression in apoptotic cell death at 24 hours reperfusion following transient forebrain ischemia.
Key W ords: IGF-I, Apoptosis, Rat, Forebrain ischemia.
( A c c e p t e d 2 9 M a r c h , 2 0 0 1 ) M a r m a r a M e d i c a l J o u r n a l 2 0 0 1 ; 1 4 ( 3 ) : 1 4 7 - 1 5 3
Correspondance to: Nazire Afşar, M. D. - Department o f Neurology, Marmara University Hospital,
INTRODUCTION
Cerebral ischemia results in both necrotic and
programmed neuronal death (1-3). The
programmed steps leading to apoptosis may be subject to modulation by extracellular conditions and thus may provide a potential for treatment in the setting of acute ischemic stroke (1,3, 4). On the other hand, neurotrophic factors and pro- and antiapoptotic proteins have been reported to modulate apoptosis (5, 6).
One of the hypotheses concerning the occurrence of apoptosis is that the death of cells may result from the loss of survival signals such as trophic factors following injury . Moreover, oxidative stress can induce apoptosis along with necrosis (7). Antioxidants are known to prevent
programmed cell death (8), whereas
neurotrophic factors have been reported to enhance neural resistance to free radical- induced damage (9) and to promote neuronal survival (10). Thus a neurotrophic factor as insulin-like growth factor (IGF-I) may provide trophic support during the critical period and prevent the induction of mechanisms of programmed cell death (10-12).
The aims of this study were to demonstrate the presence of apoptotic cell death and the pattern of expression of proapoptotic protein (bax) at different reperfusion times after transient forebrain cerebral ischemia and to observe the potential benefits of a neurotrophic factor IGF-I on these parameters. A second purpose was to
assess the correlation between these
parameters and the oxidative injury occurring during the reperfusion period.
MATERIALS A N D METHODS
Two independent studies were conducted (see experimental groups): in both arms vehicle or insulin-like growth factor I (IGF-I) was injected intracisternally after ten minutes of transient forebrain ischemia, and the histological outcome was analyzed 1 hour, 24 hours or 7 days later. The study was approved by the Institutional Ethics Committee.
Ischemia Model:
Transient forebrain ischemia was induced in adult Wistar albino rats (200 to 240 g body weight) of both sexes by bilateral carotid artery occlusion followed by hypotension (13). The animals were anesthetized by intraperitoneal injection (i.p.) of ketamine (100mg/kg) and chlorpromazine (0.75 mg/kg). Atropine (0.1 mg/kg i.p.) was used to control pulmonary and gastric secretions. The carotid arteries were exposed via a midline ventral incision and temporarily ligated with a 3/0 silk; meanwhile the femoral artery was exposed and cannulated to produce a systemic hypotension of 50mmHg (Nihon-Kohden, Model RM 6100). After ten minutes of ischemia, the blood flow in the carotid arteries was restored and blood pressure was normalized with an autologous blood transfusion. Sham-operated animals received the same anesthesia and surgery but did not undergo ischemia.
Experimental Groups:
The study comprised two arms with the same design and consisted of a transient forebrain ischemia of ten minutes, followed by three different reperfusion periods, namely 1 hour, 24 hours and 7 days. Rats in each reperfusion subgroup received either intracisternal injection using a Hamilton syringe (50 pi) of vehicle or 10 pg/rat of IGF-I, or were sham operated. Group I (n=6 for each reperfusion and sham group) was designed for the demonstration of apoptosis and bax protein expression; whereas group II (n=4 for each reperfusion group) was destined to demonstrate the degree of oxidative injury by way of measuring protein oxidation.
Fixation and Brain Processing:
Group I rats were given an overdose of ether anesthesia at one of the three prespecified reperfusion periods (see experimental groups). Rat brains were fixed by transaortal perfusion and immersion in 10% buffered formalin phosphate (14, 15). Their brains were removed and embedded in paraffin. Three consecutive coronal sections at the level of the dorsal hippocampus, each of 5 pm in thickness, were performed and were destined for hematoxylin and eosin (H&E) staining; immunohistochemistry
for bax protein expression and for a molecular biological-histochemical system specific for staining fragmented DNA and apoptotic bodies (2, 15, 16). The evaluation was performed by one of the investigators blinded to the treatment and reperfusion groups.
Group II animals were subjected to lethal doses of ether anesthesia, and following immediate decapitation brains were stored at -70“C until determination of protein oxidation.
Immunohistochemistry:
Tissue sections were deparaffinized, rehydrated, and pretreated with microwave at 750 watts for 3 minutes. Staining was performed with primary antibody raised against bax protein (Biogenex) for 30 minutes at room temperature. After washing with tris-buffered saline (TBS) slides were incubated with rabbit animal detection kit
(Biogenex) at room temperature.
Diaminobenzidine was used as chromogen after washing with TBS. Sections were washed,
counterstained with Mayer-hematoxyline,
dehydrated and mounted with aqueous mounting media.
In situ labeling of fragmented DNA:
Apoptosis was identified histologically by a pathologist using the in situ end labeling procedure for the detection of nucleosomal DNA
fragmentation, which uses the terminal
transferase reaction (TUNEL, ApopTag kit, Oncor 7100). Negative controls for the apoptotic staining consisted of consecutive sections of each case in which the tdt enzyme was omitted. Evaluation for bax protein and apoptosis expression was performed by the pathologist by light microscopy at x400 magnification by counting at least 50 neurons. The results were interpreted as percentage of neurons presenting nuclear staining among the total number scored. Protein Oxidation:
Protein oxidation was determined by reaction of the released carbonyl groups with 2,4- dinitrophenylhidrazine (DNP) yielding 2,4- dinitrophenylhidrazone, which was in turn measured by way of spectrophotometry at an absorbency of 360 nm (17). At first the tissue protein contents were measured using the
modified Lowry method (18). Brain tissue was homogenized into 20 mM K2HP04 solution (1/10 weight/volume). Ten percent streptomycin sulfate (into 50 mM Hepes, pH=7.2) was then added to the homogenate at a proportion of 1/10, and after 15 minutes was subjected to 10 minutes centrifugation at 10,000g. Thereafter, 0.5ml of 20% tri-chloroacetic acid (TCA) was added to 0.5ml supernatant. The protein precipitate thus obtained was allowed to react with 10 mM DNP (in 2 N HCL) for one hour in a dark chamber at 37°C. Proteins were then denatured a second time with 0.5ml 20% TCA. The precipitate was then washed two times with 1ml solution of ethanol : ethyl acetate (1:1) to eliminate the non reacted part of DNP. Finally proteins were diluted into 1ml 1 N NaOH and were determined by spectrophotometry at 360 nm absorbency. The values thus obtained were expressed as carbonyl amount (nmol) per mg tissue protein by using a 22,000 M'1cm'1 coefficient.
Statistical evaluation:
Kruskal-Wallis non-parametric ANOVA was used to compare protein oxidation values between
reperfusion subgroups. Logarithmic
transformation of data for apoptosis and bax expression was performed in order to allow more accurate analysis. Bax protein and apoptosis expressions between reperfusion subgroups were compared by use of a parametric ANOVA test. Parametric repeated measures ANOVA test was used to compare bax protein and apoptosis expression in IGF-I and vehicle treated animals, as well as sham operated animals within each reperfusion group. A p value < 0.05 was considered significant.
RESULTS
The results of the group I for apoptotic cell and bax protein expression are given in Table I. A significant increase in apoptotic cell expression at 24 hour reperfusion was observed in the vehicle treated group as compared to the sham operated animals (p < 0.01). Moreover, exposure to IGF-I after ischemia produced a decrease in apoptotic cell expression at 24 hour reperfusion compared to the vehicle treated group (p < 0.01), while no difference with the sham operated group
was observable (p > 0.05). Comparison of different reperfusion groups for the vehicle treated animals showed a slight increase of apoptotic staining at 24 hour reperfusion, however remaining just short of significance (p = 0.0730). In contrast to apoptosis, no difference in bax protein expression could be detected at different reperfusion times and after the use of
IGF-I as compared to the sham operated animals (p > 0.05).
Group II results for protein oxidation are given in Table II. A trend toward an increase in the amount of carbonyl group was observed with longer reperfusion periods. However no effect of IGF-I could be demonstrated (p > 0.05).
T a b l e I.: Group I, Pathology Results
Reperfusion periods Treatment (n=6) Mass (g) (mean ± SD) Apoptosis (median-% cell) Apoptosis - log (mean ± SD) Bax* (median-% cell) Bax - log (mean ± SD) 1 hour Sham 220 ± 8.8 1.0 0.46 ± 0.71 6.5 0.84 ± 0.20 Vehicle 224 ± 11.6 20.5 0.96 ± 0.79 4.0 0.67 ± 0.46 IGF-I 205 ± 19.4 1.0 0.30 ± 0.40 6.0 0.79 ± 0.32 24 hours Sham 204 ± 20.2 1.0 0.15 ± 0.37 4.5 0.69 ±0.23 Vehicle 206 ± 7.3 45.5 1.40 ± 0.69 ** 9.5 0.89 ± 0.33 IGF-I 212 ± 10.7 1.0 0.27 ± 0.54 ** 10.5 0.92 ± 0.29 7 days Sham 218 ± 18.8 2.5 0.49 ± 0.58 5.5 0.71 ± 0.27 Vehicle 212 ± 15.9 1.0 0.50 ± 0.771 10.5 0.87 ±0.40 IGF-I 211 ± 14.3 4.0 0.50 ± 0.55 4.0 0.62 ±0.27
The results are given as median percentage of apoptotic cells or bax Immunostaining cells among the total counted. * Proapoptotic protein
** p < 0.01 (vehicle vs. Sham and IGF-I vs. vehicle) t p = 0.0730
T a b l e II.: Group II, Protein Oxidation Results
Reperfusion periods Treatment (n=4) Mass (g) (mean ± SD) Carbonyl (nmol/ mg tissue protein) (median)* 1 hour Vehicle 208 ± 14.2 4.34 IGF-I 198 ± 6.9 3.50 + 24 hours Vehicle 200 ±13.3 5.36 IGF-I 192 ± 8.8 5.10 + 7 days Vehicle 214 ±17.4 6.28 IGF-I 208 ±12.6 6.13 +
* Prtoein oxidation is expressed as the amount of carbonyl group per milligram tissue protein, t non-significant.
D IS C U S S IO N
The penumbra is now widely considered as an area that is potentially salvageable, in order to prevent further enlargement of the originally infarcted area (19, 20). Studies have suggested apoptotic cell death as a mechanism of the delayed neuronal loss occurring in focal and global cerebral ischemia (1, 2, 4, 19, 21-23) . Moreover, the limitation of delayed neuronal death by the use of protein synthesis inhibitors demonstrated earlier, was further considered to be a clue to the active process occurring after ischemia (3, 24-26). DNA fragmentation, a morphological feature of apoptosis, has been demonstrated as early as 0.5 to 1 hour in focal ischemia models, increasing in extent until 24 or 48 hours (1), and persisting for up to 4 weeks (2). Whereas, in a transient forebrain ischemia model, similar to ours, apoptotic nuclear staining has been found to be prominent between 48 and 72 hours of reperfusion (23). In the present study using a transient forebrain ischemia-reperfusion model apoptosis has been demonstrated to be evident at 24 hours of reperfusion. We were thus able to show the appearance of features indicating apoptotic cell death somewhere in the first 24 hours, without being able to provide any data as to its limits in time, probably because of a too long reperfusion period maintained.
Along with the occurrence of apoptosis, the proapoptotic bax protein has been shown to be upregulated both in global ischemia (27) and focal ischemia (28, 29). The temporal profile of bax upregulation differed in various studies. Increased bax expression was associated with apoptosis and has been shown as early as 0.5 to 3 hours in global ischemia (4). It was reported to last up to 72 to 96 hours (16) following transient forebrain ischemia and from 46 hours (29) to 3 days (28) following focal ischemia.
Yet, we were unable to show an increased staining for bax protein at 1 or 24 hours or 7 days of reperfusion compared to sham animals, and thus could not correlate the occurrence of apoptosis with the expression of a proapoptotic protein. Isenmann et al (28) have recently reported the occurrence of two areas at the border of the ischemic lesion, with differential immunoreactivity for bcl-2 and bax proteins associated with apoptosis. This difference in
localization of pro- and antiapoptotic protein expression might be at the origin of the failure in the present study to correlate apoptosis with an increase in bax protein immunostaining. On the other hand Deshpande et al. (30) have postulated that cell death following transient cerebral ischemia was lacking morphological characteristics of apoptosis. Iwai et al. (31) demonstrated that DNA fragmentation occurred after structural damage of the neurons. More recently, Colbourne et al. (32) reported that electron microscopic evidence was against the presence of apoptotic cell death following global ischemia. Therefore a potential cause for the lack of correlation between apoptotic and bax protein staining observed in this study may be the absence of an apoptotic activity. However, this hypothesis can be tested with electron
microscopy testing along with the
immunostaining.
Neurotrophic factors, along with preventing apoptosis (12), may protect neurons from death by counteracting other mechanisms such as free- radical induced damage (9) or excitotoxic damage (33-35). Their action is thought to be through induction of antiapoptotic genes expression, such as bcl-2 (36), which together with preventing apoptotic cell death, were reported to act on necrotic cell death by having an antioxidant effect (35). Insulin-like growth factor-l (IGF-I) has been demonstrated to be neuroprotective when given as a continuous infusion (10) or as intracerebro-ventricular injection (12) following hypoxic-ischemic brain injury and with no effect in a transient global ischemia model (14).
In this study, we could only demonstrate a significant decrease of apoptotic cell staining with use of post-ischemic IGF-I, without any effect on bax protein expression or oxidative process following ischemia-reperfusion injury. This could either be due to the low dose of IGF-I used (1 Opig/rat), or a more dense ischemic insult induced in the present model resulting in necrotic cell death rather than apoptosis. Nevertheless, considering the encouraging results so far obtained with neurotrophic factors, and the potential to interfere with both apoptotic and necrotic cell death, we think that this field of neuroprotection merits still more research.
A C K N O W L E D G M E N T S
This study was supported by a grant from the Marmara University Research Fund number 1998/4.
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3 1. Iw a i T, fta ra A, biiwa M, e t al. T e m p o ra l p ro file o f n u c le a r DblA fra g m e n ta tio n in s itu in g e rb il h ip p o c a m p u s fo llo w in g tra n s ie n t fo re b ra in is c h e m ia . B ra in Res 1 9 9 5 ;6 7 1 :3 0 5 -3 0 8 . 3 2 . C o lb o u rn e E, S u th e rla n d GR, A u e r Rbl. E le c tro n m ic r o s c o p ic e v id e n c e a g a in s t a p o p to s is as th e m e c h a n is m o f n e u ro n a l d e a th in g lo b a l is c h e m ia . J b ie u ro s c i 1 9 9 9 ;1 9 :4 2 0 0 -4 2 1 0 . 3 3 . E in k le s te in SP, R e m m o u A, C aday CD, B e rlo v e DJ. B asic fib ro b la s t g ro w th fa c to r p ro te c ts c e re b r o c o r tic a l n e u ro n s a g a in s t e x c ita to r y a m in o a c id to x ic ity in v itro . S tro k e ¡9 9 3 :2 4 (s u p p l l ) : l - 141-1-143.
3 4 . M a ttso n MP, C heng B. G ro w th fa c to rs p ro te c t n e u ro n s a g a in s t e x c ito to x ic /is c h e m ic dam age b y s ta b iliz in g c a lc iu m h o m e o s ta s is . S tro k e 1 9 9 3 ;2 4 (s u p p l I):I-1 36-1-140.
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