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Post-ischemic treatment of pentoxifyline reduces cortical not striatal infarct volume in transient model of focal cerebral ischemia in rat
BR A I N R ES E A RC H 1 1 4 4 ( 2 00 7 ) 1 8 6 –19 1
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Post-ischemic treatment of pentoxifyline reduces cortical not striatal infarct volume in transient model of focal cerebral ischemia in rat Abedin Vakili ⁎, Mahdi Zahedi khorasani Laboratory of Cerebrovascular Research, Physiological Research Center, University of Medical Sciences, Semnan, Iran
A R T I C LE I N FO
AB S T R A C T
Article history:
Pervious studies reported that pentoxifylline (PTX) have a neuroprotective effect in the brain
Accepted 23 January 2007
trauma and the global cerebral ischemia in the experimental models. However, the effect of
Available online 1 February 2007
PTX in transient model of focal cerebral ischemia has not been investigated yet. Therefore, this study was designed to investigate the effect of post-ischemic treatment of PTX on
Keywords:
ischemic injuries in focal cerebral ischemic. Male Wistar rats (n = 32) were assigned to control
Pentoxifylline
or PTX- (60 mg/kg i.p.) treated groups. PTX at dose 60 mg/kg i.p. administered at the
Transient focal cerebral ischemia
beginning, or 1, or 3 h after ischemia. Focal cerebral ischemia was induced by middle cerebral
Rat
artery occlusion, followed by 24-h reperfusion. At the end of 24 h ischemia, neurological dysfunction score was tested and infarct volumes were determined using triphenyltetrazolium chloride staining. Administration of PTX (60 mg/kg) at the beginning of ischemia, or 1, or 3 h after ischemia significantly reduces cortical infarct volumes by 43%, 40% and 41%, respectively. However, PTX did not significantly affect striatal infarct volumes and neurological dysfunction. The findings of the present study indicate that administration of PTX at least 3 h post-transient focal stroke reduces cortical brain ischemic damage in the rat model of transient focal cerebral ischemia. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Pentoxifylline (PTX) is a drug that widely used for the treatment of intermittent claudicating, peripheral vascular and (Jacoby and Mohler, 2004) cerebrovascular disorders (Frampton and Brogden, 1995). Recent studies have reported that treatment with PTX reduces ischemic–reperfusion injuries in the lung (Thabut et al., 2001), intestine (Sener et al., 2001), liver (Iwamoto et al., 2002), kidney (Kim et al., 2001), and spinal cord (Savas et al., 2002). Moreover, PTX was shown to act
as a neuroprotectant in brain trauma (Shohami et al., 1999), global ischemia (Sirin et al., 1998) and in hypoxic–ischemic brain injury in immature rats (Eun et al., 2000). In addition, our pervious studies have shown that PTX has a neuroprotective effect when administrated at 30 min before inducing focal cerebral ischemia (Nekoeian et al., 2005). As far as the literature is concerned, almost no data about the effects of PTX on ischemic damage following transient focal cerebral ischemia are available. Therefore, the current study was conducted to investigate the effects of post-ischemic
⁎ Corresponding author. Fax: +98 231 3331551. E-mail address: [email protected] (A. Vakili). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.01.096
BR A I N R ES E A RC H 1 1 4 4 ( 2 00 7 ) 1 8 6 –1 91
Table 1 – Physiological parameters including mean arterial pressure (MAP; mm Hg), pH, PaCO2 (mm Hg), PaO2 (mm Hg) in rats receiving 1 ml/kg normal saline (as control group) or pentoxifyline (PTX, 60 mg/kg i.p.) at 10 min before and 10 min after middle cerebral artery occlusion (MCAO) Variables
pH PaCO2 Pa O 2 MAP
10 min before MCAO
10 min after MCAO
Saline
PTX
Saline
PTX
7.34 ± 0.03 46 ± 5 92 ± 8 91 ± 7
7.28 ± 0.02 43 ± 7 88 ± 9 85 ± 4
7.30 ± 0.04 45 ± 6 84 ± 10 90 ± 9
7.29 ± 0.03 47 ± 8 89 ± 14 87 ± 4
Values are means ± SEM.
treatment of PTX on cortex, striatum damage, and neurological deficit in rat model of transient focal cerebral ischemia.
at 1 or 3 h after MCAO significantly reduces infarct volume by 39% (130 ± 16 mm3, p < 0.001), 34% (140 ± 7 mm3, p < 0.001) and 30% (149 ± 10 mm3, p < 0.001), respectively (Figs. 1 and 2A). The cortical and striatal infarct volumes in control group were 156 ± 11 mm3 and 58 ± 4 mm3. Administration PTX (60 mg/kg i.p.) at the beginning, or 1 h, or 3 h after MCAO significantly reduces cortical infarct volume by 43% (88 ± 10 mm3, p < 0.001), 40% (93 ± 8 mm3, p < 0.001) and 41% (91 ± 5 mm3, p < 0.001), respectively (Fig. 2B). Moreover, PTX did not significantly affect the striatal infarct volume, regardless of when it was given (Fig. 2C and Fig. 1, p > 0.05). Moreover, PTX significantly reduced infarct area in sections of 2–6 when administrated immediately after induction of cerebral ischemia (Fig. 3). PTX-mediated neuroprotection is mainly seen in the posterior part of the MCA territory where cortical, i.e., penumbral, tissue predominates (Fig. 3).
2.3.
2.
Results
2.1.
Physiological parameters
There were no significant differences in mean arterial pressure (MAP), PaCO2, PaO2, and blood pH between control (saline, 1 ml/kg) and PTX-treated groups (Table 1).
Effect of PTX on neurological deficits score
In the saline-treated control group, the neurological deficit score was 1.86 ± 0.13 at 24 h after MCAO (Fig. 4). Administration of PTX (60 mg/kg) at the beginning (1.38 ± 0.18), at 1 h (1.29 ± 0.18), or 3 h (1.43 ± 0.20) after MCAO, did not significantly change the neurological deficits score (Fig. 4, p > 0.05).
3. 2.2.
187
Discussion
Effect of PTX on post-ischemic injuries
The total infarct volume in control group animals was 214 ± 13 mm3. Treatment with PTX (60 mg/kg i.p.) at the beginning,
The aim of this study was to evaluate the effects of postischemic treatment of PTX on ischemic damage and neurological deficit in a rat model of transient focal cerebral ischemia.
Fig. 1 – Photographs illustrating the in seven coronal sections of the rat brain with TTC staining, after 60-min MCAO and 23h reperfusion, in which red color is normal area and white color is infarct area. Colorless region corresponds to occluded MCA territory: (A) saline; (B, C, D) PTX (pentoxifyline) treated that were injected at the beginning (0 h), or at one (1 h) or three hours (3 h) after induction of ischemia. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3 – Infarct areas for 7 coronal section form anterior to posterior in rats receiving saline or PTX (60 mg/kg i.p.) at the beginning (0 h), or at one (1 h) or three hours (3 h) after induction of ischemia.
In this study, a dose of PTX (60 mg/kg) was selected, based on a pervious report and on our pervious study (Sirin et al., 1998; Nekoeian et al., 2005). Sirin and colleagues have reported that pre-ischemic treatment of PTX (60 mg/kg i.v.) significantly reduces brain injuries in transient global ischemia in rat (Sirin et al., 1998). Moreover, we have previously reported that preischemic treatment of PTX at the dose of 30 or 60 mg/kg i.p. significantly reduces infarct volume in rat model of transient focal cerebral ischemia and no significant difference between the doses of 30 and 60 mg/kg PTX on infarct volume (Nekoeian et al., 2005). On the other hand, earlier studies have shown that PTX at the doses of 10 and 20 mg/kg had no protective effect on ischemic damage in cerebral ischemia and brain trauma (Steen et al., 1982; Shohami et al., 1996). We have found that treatment with PTX given at the beginning, 1 h, or 3 h after induction ischemia resulted in the reductions of 43%, 40% and 41% in cortical infarct volumes, respectively, while they almost did not alter striatal infarct
Fig. 2 – (A) Total infarct volume, (B) cortical infarct volume, (C) striatal infarct volume in rats receiving saline or PTX (pentoxifyline, 60 mg/kg i.p.) at the beginning (0 h), or at one (1 h) or three hours (3 h) after induction of ischemia. Values are means ± SEM for each group. *Significant difference from saline group (p < 0.05). Infarct volumes are normalized for brain swelling.
Transient focal cerebral ischemia of the MCA was induced in rat by an intraluminal filament, a reliable and highly reproducible method routinely used in our laboratory (Vakili et al., 2005, 2006).
Fig. 4 – Effects of treatment with saline or PTX (60 mg/kg) on neurological deficit score at 60-min MCA occlusion and 23 h after reperfusion. PTX was injected at the beginning (0 h), or at one (1 h) or three hours (3 h) after ischemia.
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volume. This is an interesting finding of the current study, demonstrating the protective effects of PTX maintained when administered at least 3 h post-transient focal stroke. Almost no data about the effects of the post-ischemic treatment of PTX on the brain injuries following transient model of focal cerebral ischemia are available. Nevertheless, this finding of the current study is consistent with that of another report, which demonstrated that post-ischemic treatment of PTX only at a dose of 40 mg/kg i.p. reduces cortical damage by 54% and had no significant effect on striatal damage in hypoxic– ischemic injury in rat (Eun et al., 2000). Furthermore, another finding showing that infusion of PTX (60 mg/kg) 20 min before ischemia significantly reduces cerebral injuries in a rat model of transient global ischemia (Sirin et al., 1998), which may also further confirm the findings of the present study. Additionally, findings of the present study receive supports from the preclinical studies which showed that PTX and pro-pentoxifylline reduces neuronal damage following ischemia (Labs et al., 1997; Teixeira et al., 1997). Also, results of the current study showing that neuroprotection exerted by PTX was mainly seen in the cortex, which represents the penumbra, while almost no protection was observed in the infarct core, i.e., striatum. This finding is in agreement with those of pervious studies demonstrating that neuroprotective agents are less effective or even ineffective in striatum (Zhang et al., 1996; Zhang and Iadecola, 1992; Beck and Bielenberg, 1991). Perhaps the paucity of collateral anastomosis in the striatal circulation leads to more predisposes in this area to ischemia and irreversible damage (Zhang et al., 1996; Zhang and Iadecola, 1992; Ginsberg and Busto, 1989). Additionally, poor collateral blood flow might be limited by the delivery of PTX into striatum. TNF-α is a potent pro-inflammatory cytokine that is expressed and secreted at the early stage of inflammatory process (Nawashiro et al., 1997a). It has been reported that TNF-α is expressed in the ischemic neuron after focal cerebral ischemia (Liu et al., 1994). TNF-α mRNA is upregulated within 30 min in ischemic brain and reaches to peak at 6–12 h postischemia (Liu et al., 1994; Buttini et al., 1996). PTX is a phosphodiesterase inhibitor that penetrates the blood–brain barrier rapidly and efficiently after systemic administration (Watkins et al., 2003; Fujimoto et al., 1976) and inhibits TNF-α mRNA synthesis (Nataf et al., 1993; Hong et al., 1995; Hoie et al., 2004). Shohami and colleagues have reported that injection of PTX (20 mg/kg i.v.) immediately after induction of close head injury (brain trauma) significantly lowered brain TNF-α level by 80% in rat (Shohami et al., 1996). Barone and colleagues showed that the injection of exogenous TNF-α exacerbated ischemic injuries in transient or permanent MCAO (Barone et al., 1997). In addition, others' findings indicate that inhibition of TNF-α by binding proteins decreased cortical infarct volume in permanent focal cerebral ischemia in mice (Nawashiro et al., 1997a,b). On these bases, we hypothesized that at least part of the neuroprotective effects of PTX that were observed in the current study may relate to the inhibition of TNF-α synthesis. Moreover, neuroprotection exerted by PTX might be related to its ability to increase cerebral blood flow, inhibit neutrophil (Bruce et al., 1996), activate monocytes/microglias (Chao et al., 1992), attenuate the release of inflammatory mediators such as
189
platelet-activating factors (Adams et al., 1995) and prevent endothelial–leukocyte adhesion (Adams et al., 1995). Another possible mechanism of the neuroprotective action of PTX might be its ability to inhibit the generation of free radicals. It has been demonstrated that PTX protects against lipid peroxidation in in vitro and in vivo models of ischemia (Bhat and Madyastha, 2001). The neuroprotective effect of PTX that was observed in this study cannot relate to changes in arterial pressure, blood gases, and rectal temperature, because these parameters were carefully monitored and did not significantly differ among the groups studied. Despite reducing cortical infarct volumes, PTX did not improve neurological deficits. This finding demonstrated that there was no correlation between infarct volumes and neurological deficit scores. This result is consistent with that of another report, which shows no correlation between infarct size and neurological or behavioral deficits (Wahl et al., 1992). Moreover, a pervious study has reported that large decreases in infarct size are necessary to affect neurological outcome as described previously (Barone et al., 1994). As previously reported, administration of PTX, along with the reduced levels of TNF-α, improved neurological function outcomes in brain trauma (Shohami et al., 1996) and global ischemia (Sirin et al., 1998). This discrepancy might be related to differences in the model of ischemia or injury, protocols, timing and doses of drug and/or differences in functional outcome measures. In summary, the findings of the present study indicated that administration of PTX at least 3 h after ischemia reduces cortical infarct volume but did not significantly affected striatal infarct volumes and neurological deficits. We hypothesized that the neuroprotective effect of PTX might be related to the inhibition of TNF-α synthesis. This data also suggests that PTX might be useful to a stroke patient.
4.
Experimental procedures
4.1.
Animals
Male Wistar rats (Pastor Institute, Tehran, Iran) were housed in standard cages in a temperature- (22–24 °C), humidity- (40– 60%), and light period- (07:00–19:00 h) controlled environment. Experiments were performed in conformity with the university research council guidelines for conducting animal studies.
4.2.
Focal cerebral ischemia
Male Wistar rats (290–330 g) were anesthetized with chloral hydrate (400 mg/kg i.p.). Under an operation microscope, right common carotid artery (CCA) and external carotid artery (ECA) were exposed. The internal carotid artery (ICA) was also dissected to the level of petrygopalatine artery. Afterwards, a silk thread was placed loosely around the ECA stump, CCA and ECA were occluded permanently and ICA temporarily using a microvascular clip. Then a small incision was made on ECA, and a nylon thread (3–0) was inserted through. While holding the thread around ECA tightly to prevent bleeding, the microvascular clip on ICA was removed, and the nylon thread
190
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was carefully and slowly pushed forward through ICA until a light resistance was felt. Such resistance was indication that tip of nylon thread was wedged at the beginning of anterior cerebral artery (20–22 mm from CCA bifurcation), resulting in occlusion of the middle cerebral artery (Vakili et al., 2006). At 1 h after induction ischemia, the filament was slowly removed. Animals were then recovered from anesthesia, and kept in single cages for 24 h, after which neurological deficit was tested. Subsequently, animals sacrificed, and their brains were removed for determination of infarct volumes. Rectal temperature was measured by a thermometer and maintained at 37 ± 0.5 °C throughout the experiment using an electrical blanket.
4.3.
Experimental groups
Infarct volume was investigated 24 h after middle cerebral artery occlusion (MCAO) in four different groups of rats. Group 1 (n = 8) was the control group which received saline as the vehicle (1 ml/kg) at the beginning of MCAO. Groups 2–4 (each n = 8) were treatment groups which received PTX (60 mg/kg i.p.) intraperitoneally at the beginning, at 1 or 3 h after ischemia, respectively. Physiological parameters were assessed in two separate experimental groups: group 1 (n = 6) was control group which received saline (1 ml/kg) and group 2 (n = 6) was treatment group which received PTX (60 mg/kg i.p.) at the beginning of MCAO. PTX was purchased from Sigma (Germany).
4.4.
Evaluation of neurological deficits
Neurological deficits were evaluated 24 h after MCAO blindly using a five-point scoring system as described previously (Vakili et al., 2005). The scoring is as follows: 0 = normal motor function, 1 = flexion of contralateral torso or forelimb upon lifting by tail or failure to extend forepaw when suspended vertically, 2 = circling to the contralateral side but have normal posture at rest, 3 = loss of righting reflex, and 4 = no spontaneous motor activity.
4.7.
Statistical analysis
Data are presented as means ± SEM. Differences between groups were evaluated using one-way ANOVA followed by Dunnett's method or the Tukey's test as post hoc analysis to ascertain significance between groups (SigmaStat 2.0, Jandel Scientific, Erkrath, Germany). Moreover, differences between two groups were done using an unpaired t-test. Differences were considered significant at P < 0.05.
Acknowledgments This work was financially supported in part by a grant (NO: 171) from Vice Chancellor for Research, Semnan University of Medical Sciences. We are grateful to Dr. Gharemaninia of physiological research center for laboratory assistance.
Monitoring of physiological parameters
In a parallel group of animals, the left femoral artery was cannulated and the mean arterial pressure (MAP) was recorded continuously from 30 min before MCAO until 30 min after reperfusion. Arterial blood gases (ABG) were measured in 0.2 ml arterial blood samples taken 10 min before MCAO and 10 min after reperfusion.
4.5.
4.6.
Measurement of infarct volume
To calculate the infarct volume, 24 h after MCAO, rats were killed under deep anesthesia; brains were removed and immersed in 4 °C cold saline for 5 min. They were then sectioned coronally into seven 2-mm-thick slices using a Brain Matrix. Afterwards, slices were immersed in 2% triphenyltetrazolium chloride solution (Sigma, Germany), and kept at 37 °C in a water bath for 15 min. The slices were then transferred to 10% buffered formalin (Merck, Germany). About 24 h later, slices were photographed using a digital camera connected to a computer (Cannon, Japan). Infarct areas were first measured using an Image Analyzer Software (NIH Image Analyzer). The infarct volume of each slice was calculated by multiplying the infarct area of the slice by its thickness. The total infarct volume of each brain was calculated as the sum of the infarct volumes of the seven brain slices. The contribution of edema to the infarct volume was corrected by the following formula as previously described (Swanson et al., 1990): Corrected infarct volume ¼ Left hemispheresize ðRight hemispheresize Measured infarct sizeÞ:
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w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Post-ischemic treatment of pentoxifyline reduces cortical not striatal infarct volume in transient model of focal cerebral ischemia in rat Abedin Vakili ⁎, Mahdi Zahedi khorasani Laboratory of Cerebrovascular Research, Physiological Research Center, University of Medical Sciences, Semnan, Iran
A R T I C LE I N FO
AB S T R A C T
Article history:
Pervious studies reported that pentoxifylline (PTX) have a neuroprotective effect in the brain
Accepted 23 January 2007
trauma and the global cerebral ischemia in the experimental models. However, the effect of
Available online 1 February 2007
PTX in transient model of focal cerebral ischemia has not been investigated yet. Therefore, this study was designed to investigate the effect of post-ischemic treatment of PTX on
Keywords:
ischemic injuries in focal cerebral ischemic. Male Wistar rats (n = 32) were assigned to control
Pentoxifylline
or PTX- (60 mg/kg i.p.) treated groups. PTX at dose 60 mg/kg i.p. administered at the
Transient focal cerebral ischemia
beginning, or 1, or 3 h after ischemia. Focal cerebral ischemia was induced by middle cerebral
Rat
artery occlusion, followed by 24-h reperfusion. At the end of 24 h ischemia, neurological dysfunction score was tested and infarct volumes were determined using triphenyltetrazolium chloride staining. Administration of PTX (60 mg/kg) at the beginning of ischemia, or 1, or 3 h after ischemia significantly reduces cortical infarct volumes by 43%, 40% and 41%, respectively. However, PTX did not significantly affect striatal infarct volumes and neurological dysfunction. The findings of the present study indicate that administration of PTX at least 3 h post-transient focal stroke reduces cortical brain ischemic damage in the rat model of transient focal cerebral ischemia. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Pentoxifylline (PTX) is a drug that widely used for the treatment of intermittent claudicating, peripheral vascular and (Jacoby and Mohler, 2004) cerebrovascular disorders (Frampton and Brogden, 1995). Recent studies have reported that treatment with PTX reduces ischemic–reperfusion injuries in the lung (Thabut et al., 2001), intestine (Sener et al., 2001), liver (Iwamoto et al., 2002), kidney (Kim et al., 2001), and spinal cord (Savas et al., 2002). Moreover, PTX was shown to act
as a neuroprotectant in brain trauma (Shohami et al., 1999), global ischemia (Sirin et al., 1998) and in hypoxic–ischemic brain injury in immature rats (Eun et al., 2000). In addition, our pervious studies have shown that PTX has a neuroprotective effect when administrated at 30 min before inducing focal cerebral ischemia (Nekoeian et al., 2005). As far as the literature is concerned, almost no data about the effects of PTX on ischemic damage following transient focal cerebral ischemia are available. Therefore, the current study was conducted to investigate the effects of post-ischemic
⁎ Corresponding author. Fax: +98 231 3331551. E-mail address: [email protected] (A. Vakili). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.01.096
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Table 1 – Physiological parameters including mean arterial pressure (MAP; mm Hg), pH, PaCO2 (mm Hg), PaO2 (mm Hg) in rats receiving 1 ml/kg normal saline (as control group) or pentoxifyline (PTX, 60 mg/kg i.p.) at 10 min before and 10 min after middle cerebral artery occlusion (MCAO) Variables
pH PaCO2 Pa O 2 MAP
10 min before MCAO
10 min after MCAO
Saline
PTX
Saline
PTX
7.34 ± 0.03 46 ± 5 92 ± 8 91 ± 7
7.28 ± 0.02 43 ± 7 88 ± 9 85 ± 4
7.30 ± 0.04 45 ± 6 84 ± 10 90 ± 9
7.29 ± 0.03 47 ± 8 89 ± 14 87 ± 4
Values are means ± SEM.
treatment of PTX on cortex, striatum damage, and neurological deficit in rat model of transient focal cerebral ischemia.
at 1 or 3 h after MCAO significantly reduces infarct volume by 39% (130 ± 16 mm3, p < 0.001), 34% (140 ± 7 mm3, p < 0.001) and 30% (149 ± 10 mm3, p < 0.001), respectively (Figs. 1 and 2A). The cortical and striatal infarct volumes in control group were 156 ± 11 mm3 and 58 ± 4 mm3. Administration PTX (60 mg/kg i.p.) at the beginning, or 1 h, or 3 h after MCAO significantly reduces cortical infarct volume by 43% (88 ± 10 mm3, p < 0.001), 40% (93 ± 8 mm3, p < 0.001) and 41% (91 ± 5 mm3, p < 0.001), respectively (Fig. 2B). Moreover, PTX did not significantly affect the striatal infarct volume, regardless of when it was given (Fig. 2C and Fig. 1, p > 0.05). Moreover, PTX significantly reduced infarct area in sections of 2–6 when administrated immediately after induction of cerebral ischemia (Fig. 3). PTX-mediated neuroprotection is mainly seen in the posterior part of the MCA territory where cortical, i.e., penumbral, tissue predominates (Fig. 3).
2.3.
2.
Results
2.1.
Physiological parameters
There were no significant differences in mean arterial pressure (MAP), PaCO2, PaO2, and blood pH between control (saline, 1 ml/kg) and PTX-treated groups (Table 1).
Effect of PTX on neurological deficits score
In the saline-treated control group, the neurological deficit score was 1.86 ± 0.13 at 24 h after MCAO (Fig. 4). Administration of PTX (60 mg/kg) at the beginning (1.38 ± 0.18), at 1 h (1.29 ± 0.18), or 3 h (1.43 ± 0.20) after MCAO, did not significantly change the neurological deficits score (Fig. 4, p > 0.05).
3. 2.2.
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Discussion
Effect of PTX on post-ischemic injuries
The total infarct volume in control group animals was 214 ± 13 mm3. Treatment with PTX (60 mg/kg i.p.) at the beginning,
The aim of this study was to evaluate the effects of postischemic treatment of PTX on ischemic damage and neurological deficit in a rat model of transient focal cerebral ischemia.
Fig. 1 – Photographs illustrating the in seven coronal sections of the rat brain with TTC staining, after 60-min MCAO and 23h reperfusion, in which red color is normal area and white color is infarct area. Colorless region corresponds to occluded MCA territory: (A) saline; (B, C, D) PTX (pentoxifyline) treated that were injected at the beginning (0 h), or at one (1 h) or three hours (3 h) after induction of ischemia. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3 – Infarct areas for 7 coronal section form anterior to posterior in rats receiving saline or PTX (60 mg/kg i.p.) at the beginning (0 h), or at one (1 h) or three hours (3 h) after induction of ischemia.
In this study, a dose of PTX (60 mg/kg) was selected, based on a pervious report and on our pervious study (Sirin et al., 1998; Nekoeian et al., 2005). Sirin and colleagues have reported that pre-ischemic treatment of PTX (60 mg/kg i.v.) significantly reduces brain injuries in transient global ischemia in rat (Sirin et al., 1998). Moreover, we have previously reported that preischemic treatment of PTX at the dose of 30 or 60 mg/kg i.p. significantly reduces infarct volume in rat model of transient focal cerebral ischemia and no significant difference between the doses of 30 and 60 mg/kg PTX on infarct volume (Nekoeian et al., 2005). On the other hand, earlier studies have shown that PTX at the doses of 10 and 20 mg/kg had no protective effect on ischemic damage in cerebral ischemia and brain trauma (Steen et al., 1982; Shohami et al., 1996). We have found that treatment with PTX given at the beginning, 1 h, or 3 h after induction ischemia resulted in the reductions of 43%, 40% and 41% in cortical infarct volumes, respectively, while they almost did not alter striatal infarct
Fig. 2 – (A) Total infarct volume, (B) cortical infarct volume, (C) striatal infarct volume in rats receiving saline or PTX (pentoxifyline, 60 mg/kg i.p.) at the beginning (0 h), or at one (1 h) or three hours (3 h) after induction of ischemia. Values are means ± SEM for each group. *Significant difference from saline group (p < 0.05). Infarct volumes are normalized for brain swelling.
Transient focal cerebral ischemia of the MCA was induced in rat by an intraluminal filament, a reliable and highly reproducible method routinely used in our laboratory (Vakili et al., 2005, 2006).
Fig. 4 – Effects of treatment with saline or PTX (60 mg/kg) on neurological deficit score at 60-min MCA occlusion and 23 h after reperfusion. PTX was injected at the beginning (0 h), or at one (1 h) or three hours (3 h) after ischemia.
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volume. This is an interesting finding of the current study, demonstrating the protective effects of PTX maintained when administered at least 3 h post-transient focal stroke. Almost no data about the effects of the post-ischemic treatment of PTX on the brain injuries following transient model of focal cerebral ischemia are available. Nevertheless, this finding of the current study is consistent with that of another report, which demonstrated that post-ischemic treatment of PTX only at a dose of 40 mg/kg i.p. reduces cortical damage by 54% and had no significant effect on striatal damage in hypoxic– ischemic injury in rat (Eun et al., 2000). Furthermore, another finding showing that infusion of PTX (60 mg/kg) 20 min before ischemia significantly reduces cerebral injuries in a rat model of transient global ischemia (Sirin et al., 1998), which may also further confirm the findings of the present study. Additionally, findings of the present study receive supports from the preclinical studies which showed that PTX and pro-pentoxifylline reduces neuronal damage following ischemia (Labs et al., 1997; Teixeira et al., 1997). Also, results of the current study showing that neuroprotection exerted by PTX was mainly seen in the cortex, which represents the penumbra, while almost no protection was observed in the infarct core, i.e., striatum. This finding is in agreement with those of pervious studies demonstrating that neuroprotective agents are less effective or even ineffective in striatum (Zhang et al., 1996; Zhang and Iadecola, 1992; Beck and Bielenberg, 1991). Perhaps the paucity of collateral anastomosis in the striatal circulation leads to more predisposes in this area to ischemia and irreversible damage (Zhang et al., 1996; Zhang and Iadecola, 1992; Ginsberg and Busto, 1989). Additionally, poor collateral blood flow might be limited by the delivery of PTX into striatum. TNF-α is a potent pro-inflammatory cytokine that is expressed and secreted at the early stage of inflammatory process (Nawashiro et al., 1997a). It has been reported that TNF-α is expressed in the ischemic neuron after focal cerebral ischemia (Liu et al., 1994). TNF-α mRNA is upregulated within 30 min in ischemic brain and reaches to peak at 6–12 h postischemia (Liu et al., 1994; Buttini et al., 1996). PTX is a phosphodiesterase inhibitor that penetrates the blood–brain barrier rapidly and efficiently after systemic administration (Watkins et al., 2003; Fujimoto et al., 1976) and inhibits TNF-α mRNA synthesis (Nataf et al., 1993; Hong et al., 1995; Hoie et al., 2004). Shohami and colleagues have reported that injection of PTX (20 mg/kg i.v.) immediately after induction of close head injury (brain trauma) significantly lowered brain TNF-α level by 80% in rat (Shohami et al., 1996). Barone and colleagues showed that the injection of exogenous TNF-α exacerbated ischemic injuries in transient or permanent MCAO (Barone et al., 1997). In addition, others' findings indicate that inhibition of TNF-α by binding proteins decreased cortical infarct volume in permanent focal cerebral ischemia in mice (Nawashiro et al., 1997a,b). On these bases, we hypothesized that at least part of the neuroprotective effects of PTX that were observed in the current study may relate to the inhibition of TNF-α synthesis. Moreover, neuroprotection exerted by PTX might be related to its ability to increase cerebral blood flow, inhibit neutrophil (Bruce et al., 1996), activate monocytes/microglias (Chao et al., 1992), attenuate the release of inflammatory mediators such as
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platelet-activating factors (Adams et al., 1995) and prevent endothelial–leukocyte adhesion (Adams et al., 1995). Another possible mechanism of the neuroprotective action of PTX might be its ability to inhibit the generation of free radicals. It has been demonstrated that PTX protects against lipid peroxidation in in vitro and in vivo models of ischemia (Bhat and Madyastha, 2001). The neuroprotective effect of PTX that was observed in this study cannot relate to changes in arterial pressure, blood gases, and rectal temperature, because these parameters were carefully monitored and did not significantly differ among the groups studied. Despite reducing cortical infarct volumes, PTX did not improve neurological deficits. This finding demonstrated that there was no correlation between infarct volumes and neurological deficit scores. This result is consistent with that of another report, which shows no correlation between infarct size and neurological or behavioral deficits (Wahl et al., 1992). Moreover, a pervious study has reported that large decreases in infarct size are necessary to affect neurological outcome as described previously (Barone et al., 1994). As previously reported, administration of PTX, along with the reduced levels of TNF-α, improved neurological function outcomes in brain trauma (Shohami et al., 1996) and global ischemia (Sirin et al., 1998). This discrepancy might be related to differences in the model of ischemia or injury, protocols, timing and doses of drug and/or differences in functional outcome measures. In summary, the findings of the present study indicated that administration of PTX at least 3 h after ischemia reduces cortical infarct volume but did not significantly affected striatal infarct volumes and neurological deficits. We hypothesized that the neuroprotective effect of PTX might be related to the inhibition of TNF-α synthesis. This data also suggests that PTX might be useful to a stroke patient.
4.
Experimental procedures
4.1.
Animals
Male Wistar rats (Pastor Institute, Tehran, Iran) were housed in standard cages in a temperature- (22–24 °C), humidity- (40– 60%), and light period- (07:00–19:00 h) controlled environment. Experiments were performed in conformity with the university research council guidelines for conducting animal studies.
4.2.
Focal cerebral ischemia
Male Wistar rats (290–330 g) were anesthetized with chloral hydrate (400 mg/kg i.p.). Under an operation microscope, right common carotid artery (CCA) and external carotid artery (ECA) were exposed. The internal carotid artery (ICA) was also dissected to the level of petrygopalatine artery. Afterwards, a silk thread was placed loosely around the ECA stump, CCA and ECA were occluded permanently and ICA temporarily using a microvascular clip. Then a small incision was made on ECA, and a nylon thread (3–0) was inserted through. While holding the thread around ECA tightly to prevent bleeding, the microvascular clip on ICA was removed, and the nylon thread
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was carefully and slowly pushed forward through ICA until a light resistance was felt. Such resistance was indication that tip of nylon thread was wedged at the beginning of anterior cerebral artery (20–22 mm from CCA bifurcation), resulting in occlusion of the middle cerebral artery (Vakili et al., 2006). At 1 h after induction ischemia, the filament was slowly removed. Animals were then recovered from anesthesia, and kept in single cages for 24 h, after which neurological deficit was tested. Subsequently, animals sacrificed, and their brains were removed for determination of infarct volumes. Rectal temperature was measured by a thermometer and maintained at 37 ± 0.5 °C throughout the experiment using an electrical blanket.
4.3.
Experimental groups
Infarct volume was investigated 24 h after middle cerebral artery occlusion (MCAO) in four different groups of rats. Group 1 (n = 8) was the control group which received saline as the vehicle (1 ml/kg) at the beginning of MCAO. Groups 2–4 (each n = 8) were treatment groups which received PTX (60 mg/kg i.p.) intraperitoneally at the beginning, at 1 or 3 h after ischemia, respectively. Physiological parameters were assessed in two separate experimental groups: group 1 (n = 6) was control group which received saline (1 ml/kg) and group 2 (n = 6) was treatment group which received PTX (60 mg/kg i.p.) at the beginning of MCAO. PTX was purchased from Sigma (Germany).
4.4.
Evaluation of neurological deficits
Neurological deficits were evaluated 24 h after MCAO blindly using a five-point scoring system as described previously (Vakili et al., 2005). The scoring is as follows: 0 = normal motor function, 1 = flexion of contralateral torso or forelimb upon lifting by tail or failure to extend forepaw when suspended vertically, 2 = circling to the contralateral side but have normal posture at rest, 3 = loss of righting reflex, and 4 = no spontaneous motor activity.
4.7.
Statistical analysis
Data are presented as means ± SEM. Differences between groups were evaluated using one-way ANOVA followed by Dunnett's method or the Tukey's test as post hoc analysis to ascertain significance between groups (SigmaStat 2.0, Jandel Scientific, Erkrath, Germany). Moreover, differences between two groups were done using an unpaired t-test. Differences were considered significant at P < 0.05.
Acknowledgments This work was financially supported in part by a grant (NO: 171) from Vice Chancellor for Research, Semnan University of Medical Sciences. We are grateful to Dr. Gharemaninia of physiological research center for laboratory assistance.
Monitoring of physiological parameters
In a parallel group of animals, the left femoral artery was cannulated and the mean arterial pressure (MAP) was recorded continuously from 30 min before MCAO until 30 min after reperfusion. Arterial blood gases (ABG) were measured in 0.2 ml arterial blood samples taken 10 min before MCAO and 10 min after reperfusion.
4.5.
4.6.
Measurement of infarct volume
To calculate the infarct volume, 24 h after MCAO, rats were killed under deep anesthesia; brains were removed and immersed in 4 °C cold saline for 5 min. They were then sectioned coronally into seven 2-mm-thick slices using a Brain Matrix. Afterwards, slices were immersed in 2% triphenyltetrazolium chloride solution (Sigma, Germany), and kept at 37 °C in a water bath for 15 min. The slices were then transferred to 10% buffered formalin (Merck, Germany). About 24 h later, slices were photographed using a digital camera connected to a computer (Cannon, Japan). Infarct areas were first measured using an Image Analyzer Software (NIH Image Analyzer). The infarct volume of each slice was calculated by multiplying the infarct area of the slice by its thickness. The total infarct volume of each brain was calculated as the sum of the infarct volumes of the seven brain slices. The contribution of edema to the infarct volume was corrected by the following formula as previously described (Swanson et al., 1990): Corrected infarct volume ¼ Left hemispheresize ðRight hemispheresize Measured infarct sizeÞ:
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