Spermine reduces infarction and neurological deficit following a rat model of middle cerebral artery occlusion: a magnetic resonance imaging study

Neuroscience 124 (2004) 299 –304

SPERMINE REDUCES INFARCTION AND NEUROLOGICAL DEFICIT FOLLOWING A RAT MODEL OF MIDDLE CEREBRAL ARTERY OCCLUSION: A MAGNETIC RESONANCE IMAGING STUDY MD. SHIRHAN,a S. M. MOOCHHALA,b* P.-Y. NG,c J. LU,b K. C. NG,b A. L. TEO,b E. YAP,c I. NG,c P. HWANG,c T. LIM,c Y. Y. SITOH,c H. RUMPEL,d R. JOSEd AND E. LINGe

One of the biochemical processes involved in brain trauma is the synthesis and release of the natural polyamines and their subsequent effect on several targets such as the N-methyl-D-aspartate (NMDA; Ferchmin et al., 2000). Current data on the neurotoxicity of polyamines have been conflicting. Spermine, the most abundant of the polyamines, appears to be released in ischemia (Gilad et al., 1993). It has been suggested that pathological conditions are likely to enhance the neuroprotective effect of spermine (Ferchmin et al., 2000). Spermine has been reported to be neuroprotective in a gerbil model of forebrain ischemia as i.p. administration of spermine significantly decreased hippocampus and striatal cell loss (Gilad and Gilad, 1991; Gilad et al., 1993; Harada and Sugimoto, 1997). However, others have reported polyamines to be neurotoxic (Sparapani et al., 1997). Nitric oxide (NO) has been associated with both positive and negative physiological roles. The NO generated from endothelial NOS (eNOS) is critical in maintaining cerebral blood flow and reducing infarct volume. However, NO produced by both neuronal and inducible NOS has been associated with a detrimental effect (Verrecchia et al., 1995; Balkan et al., 1997; Samdani et al., 1997; Lecanu et al., 1998; Salom et al., 2000; Ding-Zhou et al., 2002). Inducible NOS (iNOS) which is not present under physiological condition can be induced shortly after ischemia and contributes to secondary late-phase damage (Samdani et al., 1997). However, most of these studies have focused on the infarct volumes while only few have looked into the neurological deficit aspect. In the present study, the neurological effects of spermine on the brain pathogenesis and neurological performance in a rat model of permanent middle cerebral artery occlusion (pMCAO) were evaluated using physiological variables, serial magnetic resonance imaging (MRI), 2,3,5,-triphenyltetrazolium chloride (TTC) staining, brain nitrate/nitrite content and neurobehavioral tests.

a Department of Pharmacology, National University of Singapore, Singapore b Defence Medical & Environmental Research Institute, DSO National Laboratories (Kent Ridge), 27 Medical Drive 09-01, Singapore 117510 c

National Neuroscience Institute, Singapore

d

Department of Radiology, Singapore General Hospital, Singapore

e

Department of Anatomy, National University of Singapore, Singapore

Abstract—The role of nitric oxide (NO) in post-ischemic cerebral infarction has been extensively examined, but few studies have investigated its role on the neurological deficit. In the present study, we investigated the effect of spermine on the temporal evolution of infarct volume, NO production and neurological deficit using magnetic resonance imaging in a model of permanent focal cerebral ischemia in rats. Spermine given at 10 mg/kg 2 h after ischemia reduced the infarct volume by 40% and abolished brain NO production and improved the neurological score 24 h, 48 h and 72 h after ischemia. Spermine also reduced the neurological deficit as evaluated by rotamex, grip strength and neurological severity score tests. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: polyamine, nitric oxide, MRI, TTC staining.

Natural polyamines, e.g. spermidine and spermine and their precursor putrescine are of considerable importance for the development and maturation of nervous system. They exhibit a number of neurophysiological and metabolic effects in the nervous system, including control of nuclei acid and protein synthesis, modulation of ionic channels, calcium-dependent transmitter release and regulation of nitric oxide synthase (NOS) and free radical scavenging (Coert et al., 2000). The polyamine system is also known to be involved in various brain pathological events such as seizure, stroke, Alzheimer’s disease and others (Berstein and Muller, 1999).

EXPERIMENTAL PROCEDURES Animals and reagents Male Sprague-Dawley rats (250 –350 g), n⫽20 for each group, were deprived of food 24 h prior to the experiment but were allowed free access to water. Group 1: sham-operated. Group 2: saline-treated with pMCAO. Group 3: spermine-treated with pMCAO. A Clinical Research Center (CRC) cocktail consisting of one part hypnorm (Jansen Pharmaceutica, Beerse, Belbica), one part dormicum (Roche, Basel, Switzerland), and two parts water was used for anesthesia. Spermine tetrahydrochloride-N,N1-Bis(3aminopropyl)-1,4-butanediamine was obtained from Sigma Chemicals (St. Louis, MO, USA). The drug was dissolved in 0.9%

*Corresponding author. Tel: ⫹65-64857201; fax: ⫹65-64857226. E-mail address: [email protected] (S. M. Moochhala). Abbreviations: ANOVA, analysis of variance; BP, blood pressure; CRC, Clinical Research Center; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; MABP, mean arterial blood pressure; MRI, magnetic resonance imaging; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; NSS, neurological severity score; pMCAO, permanent middle cerebral artery occlusion; TTC, 2,3,5,-triphenyltetrazolium chloride.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.10.050

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S. Md et al. / Neuroscience 124 (2004) 299 –304

sodium chloride solution (Sigma) and administered at 10 mg/kg. The study protocol was approved by the institutional review committee, National Neuroscience Institute and the animal welfare guidelines set forth in the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services publication 85-23, 1985) was carefully adhered to.

incubation, absorbances were measured at 540 nm by a spectrophotometer (Milton Roy, Rochester, NY, USA) and converted to nitrate/nitrite, content by comparing with a nitrate standard curve. Proteins in the supernatant were assayed by the method of Bradford (Bradford, 1976), with bovine serum albumin as a standard. Data were expressed as pmol nitrate/nitrite per mg protein.

Permanent focal cerebral ischemia

MRI analysis

The animals were anesthetized by i.p. injection of CRC cocktail (0.3 ml/l00 g body weight) and were maintained under anesthesia for the duration of the experiments. MCAO was carried out as described by Longa et al. (1989) with some modifications. Ventral midline incision was made and the pterygopalatine artery was ligated. A 4/0 suture with the tip blunted by heating was inserted via the external carotid into the internal carotid and then into the left middle cerebral artery. The ipsilateral common carotid was then ligated (Ding-Zhou et al., 2002). Spermine (10 mg/kg) or saline (control) group was i.v. infused slowly via the left femoral vein 2 h postocclusion. In the Sham-operated group, no vessel occlusion was carried out after exposure of the arteries. The animals were returned to individual cages immediately after surgery with laboratory food and drinking water ad libitum. Behavioural and physical performance tests were only carried out 24 h after surgery by which time the animals had recovered from anaesthesia. The number of animals used and their suffering was minimized.

A total of 12 animals from group 2 (MCAO, n⫽6) and group 3 (MCAO, n⫽6) were imaged 24, 48 and 72 h post-injury. MRI was performed with a Bruker 2T MRI (Ettingen, Germany) machine with custom-made rat coil. Coronal images were collected from 1.2 mm thick slices with TE 52.6 ms, TM 29.2 ms, big ⌬ 50 ms and b values of 300 and 30,000 s/cm. Lesion measurements were performed following a direct Fourier transform of the raw time domain data. Images were displayed in the coronal plane and lesions were identified as hyper-intense signals on the MRI. Selection of appropriate seed points by the operator permitted the application of a contour-tracing algorithm to delineate the boundaries of lesions. For each selected area, in each slice, pixel counts were generated. These values were then multiplied by the slice thickness and summed to yield total volume in mm3 (Lu et al., 2003). To limit subjective bias, the operator was blinded to the treatment strategy. The effect of spermine and saline on the changes in lesion volumes over time was analyzed by ANOVA with repeated measures. P-values of ⬍0.05, were considered significant.

Animal preparation The mean arterial blood pressure (MABP), arterial blood gases and pH were measured by cannulating the left carotid artery. The animal’s body temperature was monitored rectally and maintained at 36 –37 °C by a homeothermic blanket control unit (Harvard Apparatus, Oldham, England). A heparinized 24G⫻1.90 cm over-the-needle catheter (Terumo Corporation, Tokyo, Japan) was inserted into the carotid artery. MABP was monitored using a blood pressure (BP) transducer (BP TRN 050; Kent Scientific, USA, USA) and recorded by a one-channel amplifier (TRN 005; Kent Scientific), which was calibrated with a pressure transducer simulator (BP TRN 051; Kent Scientific). The BP data were recorded using a direct BP data acquisition system (DBP 001). Once the MABP was stabilized, animals were treated either with spermine or saline (control) and MABP was recorded for 2 h. Arterial blood samples were collected before treatment and 30 min, 1 and 2 h post-MCAO. A handheld blood gas/pH analyzer (Abbott Laboratories, USA) was used to analyze pH, partial pressures of arterial oxygen and carbon dioxide (PaO2 and PaCO2).

Brain nitrate/nitrite assay Brain nitrate/nitrite contents were measured 24 h (n⫽5), 48 h (n⫽5) and 72 h (n⫽5) after permanent cerebral ischemia. Infarct tissue from the brain of ischemic rats or a corresponding segment in sham-operated and non-operated rats were homogenized in 400 ␮l distilled water and centrifuged at 20,000⫻g, for 10 min, at 4 °C. Nitrates were first reduced into nitrites: 50 ␮l of supernatant were incubated for 1 h in the dark with 20 ␮l of 0.31 M potassium phosphate buffer (pH 7.5), 10 ␮l of 0.86 mM ␣-nicotinamide diphosphatase (Sigma), 10 ␮l of 0.11 mM flavine adenine dinucleotide in the presence of 20 mU nitrate reductase (Roche Diagnostics, Meylan, France). Proteins were then precipitated by adding 5 ␮l of 1 M zinc sulfate (ZnSO4; Roche Diagnostics) and samples were centrifuged (2000⫻g, 5 min, 4 °C). The total amount of tissue NO end products, nitrates plus nitrites, was then determined by Griess reaction (Green et al., 1982). Briefly, 100 ␮l of Griess reagent (1:1 mixture of 1% sulfanilamide in 5% phosphoric acid (H3PO4) and 0. 1% N-(l-naphtyl)-ethylenediamine in distilled water) was mixed with 50 ␮l of supernatant. After 10-min

TTC solution At three different time points (24, 48, 72 h, each n⫽4) after pMCAO, the animals were reanesthetized with pentobarbital and intracardially perfused with a warm (37 °C) 2% TTC solution. Their brains were quickly removed, immersed in the 37 °C TTC solution for 15 min to enhance staining and then placed in 10% buffered formaldehyde. Six serial coronal sections from each brain were cut at 2 mm intervals beginning at 3.7 mm from the bregma using a rodent brain matrix (Harvard matrix, USA).

Physical performance tests Physical performance tests were performed 24, 48 and 72 h after permanent cerebral ischemia. Rotameric and grip strength tests were performed according to our previous studies (Ng et al., 2003; Lu et al., 2003). Rotameric test was performed using a rotameric device (Columbus Instruments Rotamex 4/8 system, OH, USA) to examine the locomotory coordination ability of the animal while being placed on a rotating rod. The rotating speed of the rod was set between 5 r.p.m. (start speed) and 30 r.p.m. (end speed) for a period of 240 s. An internal micro-controller was used to detect the time at which the subject fell from the rod. The average reading (in seconds) of three successive trials was taken from each animal. Forelimb grip strength was determined using a grip strength meter (Columbus Instruments, OH, USA). An electronic digital force gauge that measured the peak force exerted by the action of the animal was used for the measurement. While being drawn along a straight line leading away from the sensor, the animal released at some point and the gauge recorded the maximum force attained. The highest reading (in Newtons) of three successive trials was taken for each animal.

Behavioral test Neurological testing was also conducted 24, 48 and 72 h after permanent cerebral ischemia. The neurological status of the rats was assessed using neurological severity score (NSS), a wellestablished scoring system (Shapiro et al., 1988). The NSS consists of tests that assess the animal’s performance in (a) inability

S. Md et al. / Neuroscience 124 (2004) 299 –304 Table 1. Effect of spermine- and saline-treated rats on physiological variables

MABP (mm Hg) Before treatment 30 min after MCAO 1 h after MCAO 2 h after MCAO PaO2 (mm Hg) Before treatment 30 min after MCAO 1 h after MCAO 2 h after MCAO PCO2 (mm Hg) Before treatment 30 min after MCAO 1 h after MCAO 2 h after MCAO pH Before treatment 30 min after MCAO 1 h after MCAO 2 h after MCAO

Saline (n⫽6)

Spermine (n⫽6)

95.8⫾6.5 96.1⫾5.4 94.2⫾7.2 97.3⫾6.1

96.4⫾3.8 97.6⫾5.1 95.6⫾4.4 96.7⫾3.5

96.6⫾5.2 99.2⫾6.4 98.5⫾4.5 99.4⫾5.3

99.4⫾6.2 97.5⫾4.7 95.3⫾3.9 98.6⫾4.1

36.2⫾4.5 35.6⫾4.1 34.8⫾5.3 35.7⫾4.7

35.7⫾3.8 35.2⫾4.2 37.1⫾3.6 36.5⫾3.2

7.35⫾0.02 7.34⫾0.04 7.32⫾0.05 7.36⫾0.07

7.32⫾0.03 7.36⫾0.04 7.35⫾0.02 7.34⫾0.05

to exit from a circle 50 cm in diameter when left in its center for 30 min or 60 min, (b) loss of righting reflex when left on its back for 30 min or 60 min or more, (c) loss of seeking behavior, (d) hemiplegia or hemiparesis. Each task is awarded with a point thus totaling up to six points. This neurological study, test on the mobility, reflexes and behavior of the rat. All the neurological tests were conducted by a single trained operator to ensure uniformity while handling these animals as well as to reduce added anxiety. A score of 0 indicates no neurological deficit while a score of six indicates the most severe impairment.

Statistical analysis The results are expressed as mean⫾S.E.M. Statistical analysis was performed using the Statistical Package for Social Science for Windows (SPSSWIN). One-way analysis of variance (ANOVA) with multiple comparison tests (LSD) was employed. The ANOVA tests were carried out for the different days for the neurological tests. Probability values less than 0.05 (P⬍0.05) were considered significant.

RESULTS

A

B

B C

Fig. 1. A series of coronal, two-weight MRI of rat brain of saline-treated at (A) 24, (B) 48, (C) 72 h after MCAO. The region of hyperintensity in the right (top-arrow tip) hemisphere of each image delineates the area of injury lesion. Notice that the total area of hyperintensity appears to increase in all the time intervals in the saline-treated rats.

MRI measurement of total infarct volumes Total infarct volumes were significantly smaller in spermine-treated rats (24 h, 48⫾6.7 mm; 48h, 52⫾9.1 mm; 72 h, 55⫾8.2 mm; P⬍0.05) when compared with saline-

MRI measures of brain damage There were no changes in T2-weighted images in shamoperated rats (not shown). Permanent occlusion of middle cerebral artery induced hemispheric infarction involving the cerebral cortex and the striatum in saline-treated rats (24, 48, 72 h; Fig. 1).

mm3

Effect of spermine on physiological variables The physiological variables measured before and after administration of spermine or saline are summarized in Table 1. Arterial blood gases, pH and mean arterial pressure remained within the physiological ranges throughout the experiment. There was no significant difference (P⬎0.05) between spermine-treated group and sham-operated controls for these parameters.

301

180 160 140 120 100 80 60 40 20 0

MRI measurement of total infarct volumes 24 hrs 48 hrs *

*

*

Spermine

72 hrs

Saline

Fig. 2. Effect of spermine on total infarct volumes induced by MCAO. Spermine was given i.v. 2 h after the onset of MCAO. Data are mean⫾S.E.M., * P⬍0.05 vs saline-treated rats.

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Brain nitrate/nitrite

pmol/mg protein

12 10

24hrs

8 6

**

*

48hrs

4

72hrs

2

in e

e

Sa l

m in

am

Sp er

N

Sh

or m

al

0

Fig. 4. Effect of spermine on nitrate/nitrite content 24, 48, 72 h after MCAO. Spermine was given i.v. 2 h after the onset of MCAO. Data are mean⫾S.E.M., * P⬍0.05 vs saline-treated rats.

Neurological and behavioral study Rotameric tests

treated rats (24 h, 74⫾6.4 mm; 48 h, 85⫾8.4 mm; 72 h, 150⫾9.7 mm; Fig. 2). Histology (TTC staining)

Grip-strength test The grip-strength score of normal rats was 9.8⫾2.1 N (24 h), 10.5⫾2.5 N (48 h), 10.1⫾2.3 N (72 h). In sham-operated rats, no significant degradation in the score was observed (24 h, 9.6⫾1.6 N; 48 h, 9.9⫾2.8 N; 72 h, 9.7⫾1.3 N). In contrast, animals subjected to MCAO and receiving

Semi-qualitative analysis of saline-treated rat brain sections showed a similar area of infarct with the MRI study at the different time points (24, 48, 72 h). Schematic brain sections from each time interval were selected at the different time intervals to show presence of infarct (Fig. 3).

250

**

200

*

24 hrs

150

48 hrs

100

72 hrs

50

al

0

N or m

Brain nitrate/nitrite content in sham-operated rats (24 h, 4⫾0.8; 48 h, 4.2⫾0.5; 72 h, 4.1⫾0.7 pmol/mg protein) was not significantly different from that of normal rats (3.5⫾0.7 pmol/mg protein) at the three time points. Nitrate/nitrite content shows a significant increase (P⬍0.05) in salinetreated rats at all three time points after MCAO (24 h, 8.2⫾0.6; 48 h, 9.7⫾0.8; 72 h, 6.8⫾0.8 pmol/mg protein). Spermine-treated rats demonstrated a significantly smaller increase in nitrate/nitrite measured at all three time points after MCAO (P⬍0.05; 24 h, 4.6⫾0.6; 48 h, 5.1⫾0.9; 72 h, 4.8⫾1.1 pmol/mg of protein; Fig. 4).

300 Seconds

Brain nitrate/nitrite studies

Rotameric performance

Sa lin e

Fig. 3. A schematic representation at each time interval for histological assessment of saline-treated rats using TTC staining (A) 24, (B) 48, (C) 72 h after MCAO. The region of hyperintensity in the right (demarcated by a black line) hemisphere of each image delineates the area of injury lesion. Notice that the area of hyperintensity appears to increase in all the time intervals in the saline-treated rats.

in e

C

Sp er m

B

Sh am

A

There was no significant neurological deficit in sham-operated (24 h, 210⫾9.8 s; 48 h, 220⫾10.8 s; 72 h, 225⫾11.2 s) as compared with normal rats (24 h, 220⫾10.2 s; 48 h, 225⫾11.3 s; 72 h, 230⫾12.4 s) as seen in their rotameric performance over the 3-day monitoring period. However, in rats subjected to MCAO and receiving only saline administration, the rotameric performance was reduced with a score of 136⫾11.8 s (24 h), 145⫾12.4 s (48 h), 152⫾13.5 s (72 h). Those administered with spermine post-injury showed a significant improvement over salinetreated animals as reflected by the improved rotameric score of (24 h, 190⫾9.9 s; 48 h, 198⫾12.4 s; 72 h, 210⫾11.7 s) in the 3-day post-injury (Fig. 5).

Fig. 5. Rotameric performance in different groups of rats. There is significant difference in rotameric performance among the groups. An improved rotameric performance is observed in the group with spermine treatment when compared with saline-treated rats (* denotes significant difference (P⬍0.05) from saline-treated rats).

S. Md et al. / Neuroscience 124 (2004) 299 –304

14 12 10 8 6 4 2 0

*

**

24 hrs 48 hrs

e

Sp

Sa

lin

in er m

am Sh

m N or

e

72 hrs

al

Newtons

Grip strength tests

Fig. 6. Grip strength in different groups of rats. Rat subjected to MCAO with spermine showed significantly higher average scores when compared with saline-treated rats (* denotes significant difference (P⬍0.05) from saline-treated rats).

saline administration showed significantly lower score of 4.4⫾1.6 N (24 h), 4.65⫾2.3 N (48 h), 4.96⫾1.5 N (72 h). The grip-strength was significantly impaired (P⬍0.05) in animals administered with spermine post-MCAO (24 h, 8.8⫾1.9 N; 48 h, 9.4⫾1.81 N; 72 h, 9.6⫾2.2 N; Fig. 6). Behavioral tests There was no significant difference in the NSS score between sham-operated rats (24 h, 0.6⫾0.24; 48 h, 0.7⫾0.13; 72 h, 0.9⫾0.22) and normal rats (24 h, 0.5⫾0.05; 48 h, 0.8⫾0.18; 72 h, 0.7⫾0.24). Following MCAO, however spermine-treated rats had significantly better NSS scores (P⬍0.05) at 24, 48 and 72 h time points (0.9⫾0.25, 1.2⫾0.24, 1.1⫾0.28) when compared with saline-treated rats (3.5⫾0.32, 4.0⫾0.36, 5.5⫾0.27; Fig. 7).

DISCUSSION In our model, pMCAO by the intraluminal suture technique induced hemispheric infarction and onset of ischemia. In order to better understand the sequelae following ischemia, various parameters were assessed with and without application of pharmacological agent, spermine. It is hypothesized that immediately following ischemia (i.e. ⬍2 h), the NO produced is via eNOS. It has been

24 hrs

48 hrs

72 hrs

e Sa l in

Sp er m in

e

* **

Sh am

N or m al

max=6

Neurological severity score 7 6 5 4 3 2 1 0

Fig. 7. NSS in different groups of rats. Spermine-treated rats showed significantly lower scores (P⬍0.05) when compared with salinetreated rats during the 3-day monitoring period (* denotes significant difference (P⬍0.05) from saline-treated rats).

303

suggested that eNOS ameliorates the adverse effect of ischemia by increasing cerebral blood flow and decreasing platelet aggregation and neutrophil adhesion (Batteur-Parmentier et al., 2000; Cooke and Dzau, 1997). In agreement with this hypothesis, previous models of transient and permanent focal cerebral ischemia had shown deleterious effects of ischemic outcomes following early intervention by NOS inhibitors (Buisson et al., 1992; Margaill et al., 1997; Kamii et al., 1996; Zhang et al., 1995). It has been hypothesized that in the late phase of ischemia (⬎2 h), however, the nitrate/nitrite content of the infarct area attributed to iNOS. This was evidenced in the study by Iadecola et al. (1996) who observed the expression of iNOS mRNA, iNOS immunoreactivity and iNOS enzyme activity in vascular and infiltrating cells 6 – 48 h after transient MCAO in rats. NO produced by iNOS has been implicated as a mediator of glutamatergic neurotoxicity acting via NMDA receptors (Balkan et al., 1997). It has been demonstrated that motor deficits produced by MCAO in both nNOS and iNOS knockout mice were less severe than in wild-type animals (Huang et al., 1994; Iadecola et al., 1997). These findings suggest that iNOS may be linked to the neurological dysfunction induced by ischemia. To evaluate the time-course of NO production, brain nitrate/nitrite was assayed in this study. This method is preferable to the ex vivo radioenzymatic assessment of NOS activity because the latter technique defines only the potential NO production in optimized conditions and this would not be a reliable index of actual NO generation in vivo. Our results show a substantial increase in NO, 24 – 48 h after MCAO, and followed by a return to near control level at 3 days. Grandati et al. (1997) also reported a similar delayed increase in brain nitrate/nitrite content in a model of transient ischemia in mice. In this study, we also looked at the effect of spermine on permanent MCAO by performing both histology and neurological function studies. This two-pronged approach is important, as studies on correlation between the infarct volume and the neurological deficit in pMCAO models appear to be lacking. There are several mechanisms by which spermine could be neuroprotective. One could be that it prevents neuronal depolarization by inhibiting synaptic transmission and NMDA-mediated Ca2⫹ currents (Ferchmin et al., 2000). Others have suggested that the antioxidant and free radical scavenging properties of spermine could be the basis of its neuroprotective activity (Ferchmin et al., 2000). Thus, increased spermine synthesis could play a role in the innate neuroprotective program. To test these hypotheses, spermine was administered (10 mg/kg) 2 h after pMCAO in this study. Our results have shown that spermine reduces ischemic infarct volume by 40% and, furthermore, it improves ischemic outcome. This is evident from the improved performance tests and neurological scores. The neuroprotective effects of spermine are unlikely to be due to the moderate and transient increase in MABP observed after its administration since comparable increase in MABP with phenylephrine was reported to have no influence on the infarct size induced by MCAO (Gursoy-

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Ozdemir et al., 2000). Our finding that spermine reduced infarct size and neurological outcome following ischemia is in agreement with a previous report showing that administration of a similar NOS inhibitor 2 h after ischemia improved neurological function in mice subjected to transient ischemia (Iadecola et al., 1997).

CONCLUSION Treatment with spermine significantly reduced infarct volumes in the brains of rats after pMCAO, as evaluated by high-resolution MRI. Histological analysis showed an increase in infarct volumes in brain ipsilateral to the injury. The location and extent of these pathologic changes correlated with MRI findings. Neurobehavioral studies showed that rotametric performance, grip-strength score and NSSs were reduced in saline-treated rats subjected to pMCAO but were significantly improved in spermine-treated rats. It is suggested that inhibition of NO at a later stage of pMCAO by spermine may represent a potential therapeutic strategy. Acknowledgements—This study was supported by National Neuroscience Institute grant NRI00/002. I would like to thank Tan Mei Ling for her technical support.

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(Accepted 29 October 2003)