Transient ischemia increases neuronal nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase co-expression in rat striatal neurons

Experimental Neurology 204 (2007) 252 – 259 www.elsevier.com/locate/yexnr

Transient ischemia increases neuronal nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase co-expression in rat striatal neurons Elisa Bizzoco, Maria-Giuliana Vannucchi, Maria-Simonetta Faussone-Pellegrini ⁎ Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Viale G. Pieraccini, 6, 50134 Florence, Italy Received 20 September 2006; revised 24 October 2006; accepted 2 November 2006 Available online 2 January 2007

Abstract In neurodegenerative diseases, an increased number of neuronal nitric oxide synthase (nNOS)-positive neurons was reported, but nothing is known on which are the neurons induced to express nNOS. Argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and nNOS act in the L-arginine–NO–L-citrulline cycle permitting a correct NO production. In the brain, nNOS-positive neurons co-expressing ASS were known, while those co-expressing ASL were not demonstrated. We investigated by immunohistochemistry the presence of these types of neurons in the rat striatum to verify whether there was a correlation between their changes due to neurotoxic insults and animal survival. Transient ischemia, a neurodegenerative insult model, was induced in rat brain by 2 h of middle cerebral artery occlusion. The striatum, the core of ischemia, was examined at 24, 72 and 144 h after reperfusion and compared with that of rats in normal condition. ASS, ASL and nNOS-positive neurons, some of the latter also expressing ASS and ASL, were present both in normal and ischemic conditions. At 24 h after reperfusion, the number of the nNOS-positive neurons and the percentage of those co-expressing ASS and ASL were significantly increased in the animals with a longer survival and at 144 h after ischemia there was an almost complete restore of the number and/or percentage of these neurons. We hypothesize that the neurons induced to express nNOS were the ASS- and ASL-positive ones and that the neurons co-expressing nNOS, ASS and ASL, since having the enzymes necessary to maintain a correct NO production, might protect from neurotoxic insults. © 2006 Elsevier Inc. All rights reserved. Keywords: Argininosuccinate lyase, ASL; Argininosuccinate synthetase, ASS; Corpus striatum; Focal transient ischemia; Neurons; Neurodegeneration; Neuroprotection; Nitric oxide, NO; L-arginine–NO–L-citrulline cycle; Neuronal nitric oxide synthase, nNOS

Introduction Nitric oxide synthase (NOS) is responsible of nitric oxide (NO) production. In nature, there are three isoforms of NOS: the neuronal NOS (nNOS) and the endothelial NOS (eNOS) are constitutive and the iNOS is inducible. nNOS and eNOS generate low quantities of NO, while iNOS produces high quantities of NO with detrimental effects on cell function. The activity of each NOS isoform is regulated by the availability of the common substrate, the aminoacid L-arginine. The brain does not express all enzymes required for L-arginine de novo synthesis; consequently, L-arginine can be obtained through the blood flow or the L-arginine–NO–L-citrulline cycle ⁎ Corresponding author. Fax: +39 055 4271385. E-mail address: [email protected] (M.-S. Faussone-Pellegrini). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.11.008

(Braissant et al., 1999). Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are the enzymes that, together with NOS, act in the L-arginine–NO–L-citrulline cycle permitting the recycling of citrulline in order to re-obtain Larginine. In particular, NOS acts on L-arginine catalyzing the synthesis of NO and citrulline, ASS, acting on citrulline, catalyzes the synthesis of argininosuccinate, ASL, cleaving argininosuccinate, reforms L-arginine (Nakamura et al., 1991). Immunohistochemical studies demonstrated that nNOS, ASS and ASL are expressed in neurons of rat brain. nNOS immunoreactivity (nNOS+) is distributed in both the perikaryon and processes of aspiny multipolar neurons (Bredt et al., 1991; Blottner et al., 1995); ASS+ and ASL+ are detected in multipolar neurons with a perikaryal and nuclear localization, and ASL+ also in glial cells (Nakamura et al., 1991; ArntRamos et al., 1992; Braissant et al., 1999; Heneka et al., 1999;

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Wiesinger, 2001). Some studies demonstrated that nNOS and ASS co-localize (Arnt-Ramos et al., 1992; Isayama et al., 1997). In the rat striatum there are two populations of neurons expressing ASS, the strongly immunoreactive magnocellular neurons and the weakly immunoreactive medium-sized neurons, only the latter is also NADPH–diaphorase positive (ArntRamos et al., 1992). Up-to-date data on ASL and nNOS colocalization are available for the spinal cord (Nakamura, 1997), sphenopalatine ganglia and cerebral perivascular nerves (Yu et al., 1997) but not for the striatum. Briefly, it is common opinion that the L-arginine–NO–L-citrulline cycle proceeds thanks to a neuro-neuronal and/or neuro-glial transport of its metabolites (Nakamura et al., 1991; Arnt-Ramos et al., 1992; Braissant et al., 1999) since the presence of neurons coexpressing nNOS and ASS or nNOS and ASL is occasional. The existence of self-sufficient neurons, co-expressing nNOS, ASS and ASL, has never been demonstrated. Several studies have postulated that the nNOS+ neurons are more resistant to the neurotoxic insults as it occurs in the brain ischemia (Thomas and Pears, 1964; Uemura et al., 1990; Morton et al., 1994) and that neurons normally nNOS-negative may initiate to express nNOS (Wu et al., 1994; Chen and AstonJones, 1994; Zhang et al., 1993). Recently, in the rat striatum after 2 h of middle cerebral artery occlusion (MCAo), we found an increase in the number of the nNOS+ neurons and a correlation between this increase and the animal survival; therefore, we hypothesized that nNOS was induced in neurons previously nNOS-negative and that the higher number of nNOS+ neurons protected the tissue from the ischemic damage (Vannucchi et al., 2005). The existence of changes in the nNOS+/ASS+ or nNOS+/ ASL+ neurons during transient ischemia has never been ascertained. In our opinion, it would be of high interest to study whether in the ischemic animals the variations in the number of the nNOS+ neurons are accompanied by changes in the number and percentage of those also expressing ASS and/or ASL and if the animal survival might be related with these changes. At this aim, we firstly ascertained whether there was a nNOS/ASL co-localization in the striatal neurons of control rats and, secondly, we evaluated by quantitative analysis the variations of these neurons and those nNOS+ and nNOS+/ ASS+ in ischemic animals with different survival and restore. Two different striatal levels representative for the anterior and posterior striatum were chosen. Materials and methods Animals Twenty-five male Wistar rats (Harlan, Italy) weighing 270– 290 g were used. They were housed in groups of three with free access to food and water and kept on a 12 h light/dark cycle. The guidelines of the European Community's Council for Animal Experiments were followed. All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals were subdivided as follows: 5 controls and 20 ischemic rats.

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Focal cerebral ischemia MCAo was induced according to the procedure by Melani et al. (1999). The studies were performed during the acute period of transient ischemia (24 h after reperfusion) and later (72 and 144 h after reperfusion). MCAo was performed to induce focal cerebral ischemia in the right hemisphere. The animals were anaesthetized with 5.0% halothane and spontaneously inhaled 1.0 to 2.0% halothane in air by use of a mask. Body core temperature was maintained at 37 °C with a recirculating pad and K module and monitored via an intrarectal type T thermocouple (Harvard, Kent, UK). The surgical procedure to occlude the MCA consisted of insertion of a 4-0 nylon monofilament (Ethilon, Johnson and Johnson, Somerville, NJ, USA), pre-coated with silicone (Xantopren, Heraeus Kulzer, Germany) and mixed with a hardener (Omnident, Germany), into the internal carotid artery via the external carotid artery to block the origin of the MCA (Zea-Longa et al., 1989). At the end of the surgical procedure, anesthesia was discontinued and the animals were allowed free access to food and water. Two hours later, the rats were newly anaesthetized with 3% halothane, and the 4-0 nylon monofilament was withdrawn. Motor behavior during the acute phase Each rat was tested soon after awakening, 10–15 min after discontinuing anesthesia, to evaluate the presence of ischemic damage. The animal was considered ischemic when, pulled by the tail, it failed to fully extend the left forepaw and turned contralaterally. Neurological evaluation (N.E.) N.E. of motor-sensory functions was carried out before surgery and at 24, 72 and 144 h after MCAo. Two examiners, blind to the procedure the rat had undergone, consecutively and independently carried out the N.E. of each animal. Adherence to a predetermined time excluded behavioral changes based on the circadian rhythm. The neurological examination consisted of six tests as developed and described by Garcia et al. (1995). The score assigned to each rat at the completion of the evaluation equaled the sum of all six-test scores: (1) spontaneous activity (score range 0–3), (2) symmetry in movement of the four limbs (score range 0–3), (3) forepaw outstretching (score range 0–3), (4) climbing (score range 1–3), (5) body proprioception (score range 1–3) and (6) response to vibrissae touch (score range 1–3). The final minimum score was 3 and the maximum 18. At 24 h after MCAo, the animals receiving a score of over 15 were considered not ischemic and were therefore discarded. Immunohistochemistry The control (n = 5) and ischemic animals at 24 h (n = 8), 72 h (n = 8) and 144 h (n = 4) after reperfusion were deeply anaesthetized and transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS) as a fixative. The

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brain was quickly removed, post-fixed overnight in 4% paraformaldehyde at 4 °C, cryoprotected in 30% sucrose in PBS for approximately 3 days at 4 °C, and then frozen in 2methylbutane (Sigma St. Louis, MO, USA) cooled on dry ice and stored at − 80 °C. Coronal cryosections of the entire brain, 30 μm thick, were collected and those located between the interaural 8.50 and 8.30 mm, between the bregma − 0.50 and − 0.70 mm (referred as anterior area) and between the interaural 7.60 and 7.20 mm, between the bregma − 1.40 and − 1.80 mm (referred as posterior area) were selected for immunohistochemistry. Double-labeling immunohistochemical analysis was done by the ‘free-floating’ technique. The sections were first pre-incubated in 5% normal goat serum (NGS) in PBS-Triton 0.3% for 40 min; then, they were incubated with the anti-rat ASS or ASL polyclonal antibodies, both raised in rabbit, a generous gift of Professor M. Mori, extensively characterized by immunohistochemistry and immunoblot analysis (Yu et al., 1995; Nagasaki et al., 1996) diluted in PBS-Triton 0,3% NGS 1% at a final concentration of 1:300 and 1:100, respectively, for 48 h at 4 °C. At the end of the incubation, sections were washed in PBS and incubated with fluorescein FITC-conjugated affinipure F(ab′)2 fragment goat anti-rabbit IgG (H + L, Jackson Immuno-Research, West Grove, PA, USA) secondary antibody, diluted 1:100 in PBS-Triton 0,3% NGS 1%, for 2 h, in the dark, at room temperature. After washes, sections were re-incubated with a monoclonal antibody against nNOS raised in mouse (Transduction Laboratories, Lexington, KY, USA, Cat no. 610308), diluted in PBS-Triton 0.3% NGS 1% at the final concentration of 1:100, over night, in the dark, at room temperature. nNOS antiserum was then revealed by using Texas Red conjugated affinity purified IgG (H + L) anti-mouse secondary antibody raised in horse (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 in PBS-Triton 0.3% NGS 1%, incubated for 2 h, in the dark, at room temperature. Sections not exposed to the primary antibodies were included as negative controls. To evaluate the specificity of the nNOS staining, a preadsorption test was also done with constitutive NOS (cNOS) rat neuronal recombinant (Calbiochem, San Diego, CA, USA) used at two different cNOS concentrations. 8 or 18 μl of cNOS (5,5 mg/ml) were added to 2 μl of nNOS undiluted antibody; the solution was incubated for 30 min at 37 °C and 30 min at room temperature, cooled at 4 °C and centrifuged in a refrigerated microfuge. Supernatant was diluted as appropriate and used immediately for control staining (see above). The best results were obtained using the larger volume of the recombinant cNOS solution. Finally, all the sections were washed in PBS, placed on gelatinized slides, covered with coverslips and mounted with Eukitt. The immunoreaction products were observed under an epifluorescence Zeiss Axioskop microscope. Immunoreaction products were also observed under a Bio-Rad 1024 confocal laser scanning microscope (Cambridge, MA, USA) with laser beam excitation at 488 and 568 nm wavelength, and 15–20 optical sections were taken at 1.5 to 1.7 μm intervals keeping all the parameters (pinhole, contrast and brightness) constant for slices from the same experiment. All images were acquired using a 60× objective. Images of 512 × 512 pixels were obtained and opened

with Confocal Assistant 4.02 and image analysis were conducted on image z-stacks which contained the entire field of interest or on one single z-plane. Images were then digitally converted to green (ASS and ASL) or red (nNOS). Quantitative analysis Ischemic and contralateral striatum volume analysis Eight coronal cryosections of the brain of rats with N.E. ≥ 12 (n = 4) and of rats with N.E. < 12 (n = 4) at 24 h after reperfusion, 30 μm thick, were collected at 0.5 mm intervals at eight different levels starting +0.50 mm from bregma to −3.00 mm from bregma. The sections were stained with acetate Cresyl Violet (1%). The volume of ischemic and contralateral striatum was measured utilizing the Scion Image analysis system and calculated in mm3. Data obtained were expressed as mean ± SEM. Student's t test or one-way ANOVA were used as appropriate by means of Microsoft Excel. Count of nNOS+/ASS+ and nNOS+/ASL+ neurons It was performed on 2 adjacent sections taken from the two levels of the striatum of each rat (5 controls, 8 ischemic rats at 24 h, 8 at 72 h and 4 at 144 h after reperfusion). All nNOS/ASS and nNOS/ASL labeled sections were examined under fluorescence microscope at 20× magnification, and an average of 32 optical fields for the anterior area and of 18 optical fields for the posterior area (area of each optical field: 0.3 mm2) were collected using AxioVision 4. Field edges were defined based on structural details within the tissue section to ensure that the fields did not overlap. For each section, the total number of the nNOS+/ASS+ and nNOS+/ASL+ neurons and the total number of nNOS+ neurons were evaluated. All data obtained were expressed as mean ± SEM. Student's t test or one-way ANOVA were used as appropriate by means of Microsoft Excel. Differences at p < 0.05 were considered to be statistically significant. Results Neurological evaluation (N.E.) All the animals that survived at 24 h were evaluated for their motor–sensory functions and a score was attributed to each of them. At this time-point, the distribution of the scores allowed for dividing the ischemic animals into two groups: the first group of rats was characterized by a score ranging from 12 to 15 (n = 4 at 24 h, n = 4 at 72 h and n = 4 at 144 h) and the second group of rats (n = 4 at 24 h and n = 4 at 72 h) by a score ranging from 4 to 11. The time-course of motor-sensory functions of the first group of animals had a little trend to recovery at 144 h from reperfusion; the second group was not characterized by a recovery and none of the rats was alive at 144 h. Infarct volume A significant increase in the volume (mm3) of the ischemic hemisphere compared with the contralateral was detected either in the animals with higher score (134.1 ± 1.0 vs. 122.4 ± 1.2,

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Table 1 Rats

Contralateral striatum (mm3)

Ischemic striatum (mm3)

N.E. ≥ 12 N.E. < 12

23.25 ± 0.8 22.26 ± 0.1

27.9 ± 2.0 33.4 ± 1.0*°

Striatal volume of the contralateral and ischemic hemispheres of rats underwent to transient MCAo. Striatal volume is expressed in mm3. The values are expressed as mean ± SEM (n = 4 each group of animals). *p < 0.001 significantly different from the contralateral striatum. °p < 0.006 significantly different from the ischemic striatum of the rats with N.E. ≥ 12.

p < 0.006) or in those with lower score (152.1 ± 0.2 vs. 122.0 ± 2.1, p < 0.001). Moreover, the volume of the ischemic hemisphere of the rats with the lower score was significantly larger (p < 0.008) in comparison to the rats with the higher score. This difference was due to a more extended edema in the former group of rats. The volumes of the ischemic and contralateral striatum of the two groups of rats are shown in Table 1. Immunohistochemistry Control and ischemic animals nNOS+, ASS+ and ASL+ neurons were present at both levels examined. The nNOS+ neurons were scattered at both levels and were medium-sized (12–15 μm). All of them had a triangular or ovoid cell body with 2–3 main long processes and immunoreactivity was uniformly distributed in both soma and processes (Fig. 1A). The ASS+ neurons (Figs. 1B and C) were numerous and everywhere distributed. They were of either medium (12– 15 μm) or large size (25–30 μm) and in all of these cells the labeling appeared as immunoreactive granules distributed in the nucleus, the perikaryon and the main processes. The nuclear granules were larger and more intensely labeled than the cytoplasmatic ones and the nucleolus was always unlabeled. The medium-sized ASS+ neurons had an ovoid body provided with 2–3 main processes (Fig. 1B); the large-sized ASS+ neurons had a polygonal body provided with 4 or more main processes (Fig. 1C). Furthermore, the cytoplasmatic labeling of these neurons

Fig. 2. Striatum of control rats. Double labeled neurons. In green, the ASS and ASL immunoreactivity (ASS+ and ASL+), in red the NOS immunoreactivity (nNOS+) in yellow the nNOS+/ASS+ and nNOS+/ASL+ labeling. (A) One nNOS+/ASS+ neuron with a mixture of red and yellow labeling in the cytoplasm. The nucleus is ASS+ (green). Processes far from the cell body are nNOS+ (red). (B) One nNOS+/ASL+ neuron with a mixture of red and yellow labeling in the cytoplasm. The nucleus is ASL+ (green). Processes are nNOS+ (red). Fluorescent microscope. Scale bar: A and B = 15 μm.

Fig. 1. Striatum of control rats. In red the NOS immunoreactivity (nNOS+) and in green the ASS and ASL immunoreactivity (ASS+ and ASL+). (A) Two nNOS+ neurons. (B) One ASS+ neuron of medium size with an intensely labeled nucleus and moderately labeled perikaryon and processes. (C) One ASS+ neuron of large size with intensely labeled nucleus, perikaryon and processes. (D) Numerous ASL+ cells. The nuclear labeling is intense and the cytoplasmatic one is weak. The cells surely identifiable as neurons (arrows) are those with the cytoplasm being labeled also at the first portion of the processes, and are medium-sized. Confocal microscope. Scale bar: A–D = 25 μm.

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was stronger than that of the medium-sized neurons. The ASL+ neurons (Fig. 1D) were very numerous and everywhere distributed. All of them had a medium size (12–15 μm), an ovoid–shaped body provided of 2–3 main processes. Similarly to the ASS+ neurons, the nuclear labeling was granular and strong and the nucleolus was unlabeled. Differently from the ASS+ neurons, the cytoplasmatic labeling appeared as small particles and only present in the perikaryon and at the beginning of the main processes. There were also some more ASL+ cells whose processes were not labeled and, therefore, not identifiable with certainty as neurons. nNOS+/ASS+ (Figs. 2A, 3A) and nNOS+/ASL+ neurons (Figs. 2B, 3D) were also present. All these neurons were medium-sized and appeared as ovoidtriangular cells with 2–3 main processes. nNOS+, nNOS+/ASS+, nNOS+/ASL+, ASS+ and ASL+ neurons were also present in all the ischemic animals. Their size and labeling distribution and intensity were similar to controls (Figs. 3A–F). Quantitative analysis Control animals Quantitative analysis demonstrated that at both striatal levels examined either the number and the percentage of the nNOS+

neurons co-expressing ASS (nNOS+/ASS+ neurons) were significantly lower than those of the nNOS+ neurons coexpressing ASL (nNOS+/ASL+ neurons) (Figs. 4A–D; Table 2). Ischemic animals By quantitative analysis, the number of the nNOS+ neurons and the number and/or percentage of the nNOS+/ASS+ and nNOS+/ASL+ neurons were different from controls, either in the ischemic rats with N.E. ≥ 12 or in those with N.E. < 12, at both striatal levels (Figs. 4A–D; Table 2). Animals with N.E. ≥ 12. At both striatal levels, the number of the nNOS+, nNOS+/ASS+ and nNOS+/ASL+ neurons was significantly increased at 24 h after reperfusion. Conversely, the number of all these neurons was significantly decreased at 72 h if compared to that at 24 h and, except for the nNOS+/ASS+ neurons, also respect to controls. At 144 h, the number of the nNOS+ and nNOS+/ASS+ neurons was similar to controls. Moreover, the number of the nNOS+/ASL+ neurons was significantly higher than at 72 h after reperfusion at both striatal levels. Finally, to be noticed that at 24 and 72 h after reperfusion, the percentage of the nNOS+/ASL+ neurons remained similar to that of controls at both levels considered, while the percentage of the nNOS+/ASS+ neurons was

Fig. 3. Striatum of control and ischemic rats. Double labeled neurons. In green, the ASS and ASL immunoreactivity (ASS+ and ASL+), in red the NOS immunoreactivity (nNOS+) in yellow the nNOS+/ASS+ and nNOS+/ASL+ labeling. (A–C) ASS+ neurons (green), nNOS+ neurons (red) and nNOS+/ASS+ neurons (with a mixture of red, green and yellow labeling in the cytoplasm). (A) control rats. (B and C) ischemic rats at 24 h (B) and 144 h (C) after reperfusion and with N.E. ≥ 12. (D–F) ASL+ neurons (green), nNOS+ neurons (red), nNOS+/ASL+ neurons (with a mixture of red, green and yellow labeling in the cytoplasm). (D) Control rats. (E and F) Ischemic rats at 24 h (E) and 144 h (F) after reperfusion and with N.E. ≥ 12. Fluorescent microscope. Scale bar: A–F = 50 μm.

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Fig. 4. Quantitative analysis. (A and B) Anterior striatum; (C and D) posterior striatum. Number of the nNOS+ neurons expressing ASS (nNOS+/ASS+) (A and C ■) and do not expressing ASS (nNOS+/ASS−) (A and C □) and number of the nNOS+ neurons expressing ASL (nNOS+/ASL+) (B and D ) and do not expressing ASL (nNOS+/ASL−) (B and D □) in the anterior and posterior striatum of ischemic rats with N.E. ≥ 12 at 24, 72 and 144 h and with N.E. < 12 at 24 and 72 h after reperfusion. In the anterior (A) and posterior (C) striatum of rats with N.E. ≥ 12, the number of nNOS+/ASS+ neurons is significantly increased at 24 h respect to controls, significantly decreased at 72 h respect to 24 h, similar to controls at both 72 and 144 h. In the anterior striatum of rats with N.E. < 12, the number of the nNOS+/ASS+ neurons is significantly increased at 24 h and returns similar to controls at 72 h. In the posterior striatum of rats with N.E. < 12, the number of the nNOS+/ASS+ neurons at 24 and 72 h is similar to controls. The number of the nNOS+/ASS− neurons is significantly decreased at 24 and 72 h in both groups of ischemic rats, either in the anterior and posterior striatum, and returned similar to controls at 144 h in the rats with N.E. ≥ 12. In the anterior (B) and posterior (D) striatum of rats with N.E. ≥ 12, the number of the nNOS+/ASL+ neurons is significantly increased at 24 h respect to controls, significantly decreased at 72 h respect to controls and 24 h; significantly increased at 144 h respect to 72 h. In the anterior striatum of rats with N.E. < 12, the number of the nNOS+/ASL+ neurons is significantly decreased at both 24 and 72 h. In the posterior striatum of rats with N.E. < 12, the number of the nNOS+/ASL+ neurons is significantly decreased only at 72 h respect to controls. In the anterior and posterior striatum of both groups of ischemic rats the number of the nNOS+/ASL− neurons is similar to controls at 24 h and significantly decreased at 72 h. At 144 h, the number of the nNOS+/ASL− neurons is significantly increased in the anterior striatum and similar to controls in the posterior striatum. nNOS+/ASS+ and nNOS+/ASL+ neurons: °Significantly different vs. control rats; °°Significantly different vs. 24 h; °°°Significantly different vs. 24 and 72 h. nNOS+/ASS− and nNOS+/ASL− neurons: *Significantly different vs. control rats; **Significantly different vs. 24 h. The differences were considered statistically significant for a p < 0.05.

significantly increased, up to reach a percentage similar to that of the nNOS+/ASL+ neurons. At 144 h after reperfusion, the percentage of the nNOS+/ASL+ neurons was significantly decreased in the anterior striatum. Animals with N.E. < 12. At both striatal levels, the number of the nNOS+ neurons was significantly reduced either at 24 or 72 h after reperfusion. The number of the nNOS+/ASS+ neurons was increased and that of the nNOS+/ASL+ neurons decreased at 24 and 72 h after reperfusion, but their percentages were high and similar to those found in the animals with N.E. ≥ 12. Discussion The present study shows for the first time that transient ischemia induces changes in nNOS, ASS and ASL co-

expression in the rat striatal neurons. Indeed, by quantitative analysis, the number and percentage of the nNOS+, nNOS+/ ASS+ and nNOS+/ASL+ neurons were evaluated either in control rats or in rats which underwent 2 h of occlusion of MCA and significant changes were seen at different time-points after reperfusion. The significantly increased number of the nNOS+ neurons found at 24 h after reperfusion in the rats with a longer survival shows that a nNOS induction has occurred in these animals and this increase is associated with reduced tissue damage. Moreover, the significant increase at this same time point in number and percentage of the nNOS+ neurons coexpressing ASS or ASL suggests that these populations of nNOS+ neurons are those more resistant to the neurotoxic insult and, likely, those able to protect from the ischemic damage. We presently found that size, shape and labeling distribution and intensity of the ASS+ and ASL+ neurons were similar to

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Table 2 Rats

Control Ischemic N.E. ≥ 12

Ischemic N.E. < 12

Time % nNOS+/ASS+ points Anterior Posterior 24 h 72 h 144 h 24 h 72 h

32.1 ± 3.1° 43.05 ± 4.3°° 83.6 ± 2.8* 81.1 ± 3.3* 97.4 ± 1.1* 97.9 ± 1.2* 33.0 ± 3.8 59.1 ± 5.9 66.7 ± 3.3* 70.1 ± 4.8* 95.2 ± 1.5* 84.2 ± 3.8*

% nNOS+/ASL+ Anterior

Posterior

83.0 ± 2.3 92.3 ± 1.9 91.1 ± 2.0 50.8 ± 3.8* 73.8 ± 2.8 91.4 ± 2.4

87.9 ± 2.5 92.0 ± 2.2 86.1 ± 4.6 80.3 ± 3.4 83.8 ± 3.1 88.9 ± 3.3

Percentages (mean ± SEM) of the nNOS+/ASS+ and nNOS+/ASL+ neurons on the total number of the nNOS+ neurons in the anterior and posterior striatum of control rats (n = 5) and ischemic rats with N.E. ≥ 12 and N.E. < 12 at 24 h (n = 4 for each group), 72 h (n = 4 for each group) and 144 h (n = 4) after reperfusion. *Significantly different versus control rats; °nNOS+/ASS+ significantly different versus nNOS+/ASL+ in the anterior striatum; °°nNOS+/ASS+ significantly different versus nNOS+/ASL+ in the posterior striatum. The differences were considered statistically significant for a p < 0.002.

those reported in the literature (Nakamura et al., 1991; ArntRamos et al., 1992; Nakamura, 1997; Heneka et al., 1999) and that, as already reported (Arnt-Ramos et al., 1992), the nNOS+/ ASS+ neurons were medium-sized cells. Moreover, we demonstrated for the first time in the striatum the presence of nNOS+/ASL+ neurons, whose size and shape are similar to those of the nNOS+/ASS+ neurons and, by confocal microscope examination, we could observe in detail the intracellular distribution of both ASS+ and ASL+. Briefly, according to our results, three populations of neurons able to produce NO are present in the rat striatum: the neurons expressing only nNOS (that we called nNOS+/ASS− and nNOS+/ASL− neurons in Fig. 4), the nNOS+/ASS+ and the nNOS+/ASL+ neurons. Moreover, there are also ASS+ and ASL+ neurons, both involved in the L-arginine–NO–Lcitrulline cycle but not able to produce NO. The antibodies we used did not allow to verify whether all or some of the nNOS+ neurons co-expressed ASS and ASL, i.e. whether there were also nNOS+/ASS+/ASL+ neurons, and, similarly, whether there were ASS+ neurons co-expressing ASL, i.e. ASS+/ASL+ neurons. The existence of nNOS+/ASS+/ASL+ and of ASS+/ASL+ neurons has never been, and at present cannot be demonstrated either by using these same or the other available antibodies; therefore, these possibilities can be neither excluded nor confirmed. The quantitative evaluation demonstrated that at all striatal levels the percentage of the nNOS+ neurons co-expressing ASS was low, in agreement with ArntRamos et al. (1992), and that of the nNOS+/ASL+ neurons was significantly higher. Therefore, it is very likely that some of the nNOS+/ASL+ neurons do not co-express also ASS; however, it cannot be excluded that a discrete percentage of these neurons might also be ASS+, thus allowing us to postulate the existence of nNOS+/ASS+/ASL+ neurons. In summary, on the basis of our data and those of literature (Nakamura et al., 1991; ArntRamos et al., 1992; Braissant et al., 1999), we can conclude that in the rat striatum there are several populations of neurons able to produce NO, many of which needing to exchange the metabolites involved in the L-arginine–NO–L-citrulline cycle and, possibly, one population of self-sufficient neurons, the nNOS+/ASS+/ASL+ neurons.

In the ischemic rats, nNOS+, nNOS+/ASS+, nNOS+/ASL+ as well as ASS+ and ASL+ neurons were present at all timepoints studied. However, quantitative analysis revealed important changes in the nNOS+ neuronal populations in these animals at both striatal levels. The total number of the nNOS+ neurons was significantly increased after 24 h of reperfusion in the rats with better motor-sensory performances and a longer survival and reduced in those animals with the worst motor–sensory performances and not alive at 144 h after reperfusion. An increase in the number of the nNOS+ neurons was already reported for the ischemic striatum of animals with the best motor–sensory performances and interpreted as sign of protection from the damage (Vannucchi et al., 2005). It is known that at nonsaturating L-arginine concentration nNOS produces low concentrations of NO together with reactive oxygen species that, interacting, generate cellular peroxinitrite responsible for the tissue damage (Stuehr, 2004). It is possible that in the oedematous tissue of the ischemic brain the L-arginine concentration is reduced in the intercellular space; therefore, the nNOS+ neurons expressing also ASS and ASL are those neurons that should be able to maintain the cellular saturating L-arginine concentration thus allowing an adequate production of NO. In this regard, to have found that the number of the nNOS+/ASS+ and nNOS+/ASL+ neurons was significantly higher at 24 h after reperfusion than in controls and that the percentage of the nNOS+/ASS+ neurons was become so high as that of the nNOS+/ASL+ neurons fits well with the possibility that these neurons are able to maintain the NO synthesis at a correct final concentration. The increased number of the nNOS+ neurons is considered due to a nNOS induction in cells that in normal animals are nNOS-negative (Wu et al., 1994; Chen and Aston-Jones, 1994; Zhang et al., 1993, Vannucchi et al., 2005); which is the population of the neurons that can be induced to express nNOS, however, has not yet been identified. The increase in the nNOS+ neurons found at 24 h correlated with an increasing number of those co-expressing ASS and ASL; therefore, it seems reasonable to hypothesize that nNOS induction has occurred in the ASS+ and ASL+ neurons, which are very numerous in the striatum. Such an induction could also occur in the ASS+/ASL+ neurons, if present, as well as a co-induction of nNOS and ASS and of nNOS and ASL might also occur. In this regard, literature refers that ASS, ASL and the inducible isoform of NOS can be induced and also co-induced in neurons, glial cells and other cell types after cytokine treatment (Zhang et al., 2000; Nagasaki et al., 1996; Haas et al., 2002; Hattori et al., 1994; Nussler et al., 1994; Heneka et al., 1999). Briefly, an induction and/or coinduction could cause the appearance or increase the number of self-sufficient nNOS+/ASS+/ASL+ neurons. At 72 h after reperfusion, the critical time-point for both groups of ischemic rats, practically all of the nNOS+ neurons co-expressed ASS or ASL. The persistence at this time-point of the neuronal populations able to maintain a cellular saturating Larginine concentration and likely an autonomic production of NO is a proof in favor for a major resistance to neurotoxic insults of these types of nNOS+ neurons. A restore of the control picture was observed at both levels of the striatum in the rats still alive at 144 h after reperfusion,

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although it was not yet complete. Interestingly, as a common feature for all the animals surviving at 144 h after reperfusion and at both striatal levels, there was an increase in the number of the nNOS+ neurons respect to that at 72 h after reperfusion, thus indicating that these cells were alive at 72 h. In conclusion, we demonstrate that transient ischemia induces in the rat striatum significant changes in the number and percentage of the nNOS+ neurons also expressing ASS or ASL. In particular, at 24 h after reperfusion in the animals with a longer survival and better motor-sensory performances there was an increase in these neuronal populations in parallel with an increase of the nNOS+ neurons. These findings allow us to hypothesize that the nNOS+/ASS+ and the nNOS+/ASL+ neurons are resistant to ischemia and, since carrying at least two of the three enzymes of the L-arginine–NO–L-citrulline cycle, protective from the tissue damage. Acknowledgments This work was supported by a MIUR 40% grant and University of Florence ex-60% funds. We wish to thank Professor Masataka Mori for having done us the ASS and ASL antibodies and Dr. Letizia Corsani for her precious technical help. References Arnt-Ramos, L.R., O'Brien, W.E., Vincent, S.R., 1992. Immunohistochemical localization of argininosuccinate synthetase in the rat brain in relation to nitric oxide synthase-containing neurons. Neuroscience 51, 773–789. Blottner, D., Grozdanovic, Z., Gossrau, R., 1995. Histochemistry of nitric oxide synthase in the nervous system. Histochem. J. 27, 785–811. Braissant, O., Gotoh, T., Loup, M., Mori, M., Bachmann, C., 1999. L-arginine uptake, the citrulline-NO cycle and arginase II in the rat brain: an in situ hybridization study. Mol. Brain Res. 70, 231–241. Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M., Snyder, S.H., 1991. Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 7, 615–624. Chen, S., Aston-Jones, G., 1994. Cerebellar injury induces NADPH diaphorase in Purkinje and inferior olivary neurons in the rat. Exp. Neurol. 126, 270–276. Garcia, J.H., Wagner, S., Liu, K.F., Hu, X.J., 1995. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 26, 627–634. Haas, J., Storch-Hagenlocher, B., Biessmann, A., Wildemann, B., 2002. Inducible nitric oxide synthase and argininosuccinate synthetase: coinduction in brain tissue of patients with Alzheimer's dementia and following stimulation with beta-amyloid 1–42 in vitro. Neurosci. Lett. 322, 121–125. Hattori, Y., Campbell, E.B., Gross, S.S., 1994. Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis. J. Biol. Chem. 269, 9405–9408. Heneka, M.T., Schmidlin, A., Wiesinger, H., 1999. Induction of argininosucci-

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