Abstract

In view of the ability of neurotensin (NT) to increase glutamate release, the role of NT receptor mechanisms in oxygen–glucose deprivation (OGD)–induced neuronal degeneration in cortical cultures has been evaluated by measuring lactate dehydrogenase (LDH) levels, mitochondrial dehydrogenase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide levels, and microtubule-associated protein 2 (MAP2) immunoreactivity. Apoptotic nerve cell death was analyzed measuring chromatin condensation with Hoechst 33258, annexin V staining, and caspase-3 activity. Furthermore, the involvement of glutamate excitotoxicity in the neurodegeneration-enhancing actions of NT was analyzed by measurement of extracellular glutamate levels. NT enhanced the OGD-induced increase of LDH, endogenous extracellular glutamate levels, and apoptotic nerve cell death. In addition, the peptide enhanced the OGD-induced loss of mitochondrial functionality and increase of MAP2 aggregations. These effects were blocked by the neurotensin receptor 1 (NTR1) antagonist SR48692. Unexpectedly, the antagonist at 100 nM counteracted not only the NT effects but also some OGD-induced biochemical and morphological alterations. These results suggest that NTR1 receptors may participate in neurodegenerative events induced by OGD in cortical cultures, used as an in vitro model of cortical ischemia. The NTR1 receptor antagonists could provide a new tool to explore the clinical possibilities and thus to move from chemical compound to effective drug.

Introduction

The brain requires a continuous supply of oxygen and glucose to maintain normal function and viability. The idea that hypoxic–ischemic brain damage can be explained by extracellular accumulation of glutamate and overstimulation of glutamate receptors is appealing. In particular, N-methyl-D-aspartate (NMDA) receptors (Goldberg and Choi 1993; Zipfel et al. 2000) may contribute to hypoxic–ischemic neuronal injury, and in fact, treatment with glutamate antagonists can limit hypoxic–ischemic brain damage (Mueller et al. 1999; Johnston et al. 2001; Culmsee et al. 2004). Many aspects of ischemic neurodegeneration have been demonstrated in animal models. However, the ischemic process represents a complex environment for the dissection of the cellular and molecular mechanisms involved in ischemic neurodegeneration (Arundine and Tymianski 2004; Young et al. 2004). Cell culture systems could therefore represent a more defined microenvironment and a simple experimental model to study some aspects of ischemic-induced neurodegeneration (Choi et al. 1990). It has been demonstrated in cultured cortical neurons that glutamate excitotoxicity via NMDA receptors induces apoptosis or necrosis depending on the intensity of the insult. Indeed, a mild glutamate insult leads to an apoptotic cell death, whereas an intense glutamate insult induces predominantly a necrotic process (Cheung et al. 1998). Therefore, endogenous compounds able to modulate glutamatergic transmission may interfere with glutamate-induced cell death.

In this context, it has been shown that the tridecapeptide neurotensin (NT) significantly enhances glutamatergic signaling in both in vitro (Ferraro et al. 2000; Matsuyama et al. 2003; Chen et al. 2006) and in vivo (Ferraro et al. 1995, 1998, 2001) studies. These findings suggest a reinforcing action of NT on several functions exerted by glutamate in the central nervous system, in particular on the glutamate-mediated excitotoxicity (Antonelli et al. 2007). An involvement of NT in modulating glutamate excitotoxicity has recently been demonstrated in primary cultures of mesencephalic dopamine (Antonelli et al. 2002) and cortical (Antonelli et al. 2004) neurons. Nevertheless, the effects of NT on glutamate transmission in the cerebral cortex, an important cerebral area damaged by pathological events like ischemia, are still undefined. In this context, an increase of NT levels in rat cerebral cortical areas has been demonstrated following focal ischemia by Allen et al. (1995), suggesting a possible involvement of NT in ischemic brain damage. Furthermore, in vivo experiments provide evidence that NT-induced hypothermia improves neurologic outcome and reduces infarct volume after hypoxic ischemia (Katz et al. 2004) and middle cerebral ischemia (Torup et al. 2003), respectively.

In view of the neuroprotection caused by NT-induced hypothermia (Kokko et al. 2005) in in vivo models, the aim of the present study was to investigate under normothermic conditions the role of neurotensin receptor 1 (NTR1) in nerve cell death and endogenous extracellular glutamate levels after oxygen–glucose deprivation (OGD) in cerebral cortex cell cultures using a selective NTR1 antagonist SR48692. This in vitro approach may provide a more defined microenvironment where the presence of a vascular compartment and changes in temperature do not influence the results obtained.

Materials and Methods

Primary Cultures of Rat Cortical Neurons

Primary cultures of cortical neurons were prepared from embryonic day 18 Sprague–Dawley rats. Removed cortices were dissected free of meninges and dissociated in 0.025% (w/v) trypsin. The tissue fragments were dissociated mechanically by repeated gentle pipetting through wide- and narrow-bore fire-polished Pasteur pipettes in culture medium (Neurobasal medium with supplements of 0.1 mM glutamine, 10 μg/ml gentamicina, and 2% B27). The cells were counted and then plated on poly-L-lysine (5 μg/ml)–coated dishes at a density of 2.5 × 106 cells per dish and on poly-L-lysine (5 μg/ml)–coated multiwells (24 wells) at a density of 200 000 cells per well. In the dishes used for Hoechst 33258 nuclear staining, annexin V staining, and microtubule-associated protein 2 (MAP2) immunocytochemistry (see below), the cells were plated on poly-L-lysine (50 μg/ml)–coated glass coverslips. Cultures were grown at 37 °C in a humidified atmosphere of 5% CO2/95% air. Cytosine arabinoside (1 μM) was added at 5 days in vitro (DIV) to prevent glial cell proliferation. The cultures were maintained for 8 DIV before experiments.

OGD Exposure

The cultures were exposed to a transient OGD. To this purpose, in the OGD group, the culture medium was replaced with a glucose-free Krebs–Ringer bicarbonate buffer that had previously been saturated with 95% N2/5% CO2 and heated to 37 °C. The cultures were then put into an anaerobic incubator (pO2 < 2 mm Hg) with an atmosphere of 95% N2 and 5% CO2 and 98% humidity at 37 °C for 60 min. OGD was terminated by removing the cultures from the anaerobic incubator, by replacing the exposure buffer with oxygenated Krebs–Ringer bicarbonate buffer containing glucose and returning the cultures to the incubator under normoxic conditions. In control group, not submitted to OGD, the cultures were exposed to oxygenated Krebs–Ringer bicarbonate buffer containing glucose and placed in a humidified atmosphere of 5% CO2/95% air for 60 min. All the experiments were performed 24 h later.

Determination of Endogenous Extracellular Glutamate Levels

On the day of the experiment, the cells were rinsed twice by replacing the culture medium with Krebs–Ringer bicarbonate buffer (37 °C). Thereafter, 3 consecutive fractions were collected renewing this solution (400 μl). The first 2 samples, collected every 50 min (before OGD), have been used to assess basal endogenous glutamate levels, whereas the third fraction was collected 24 h later. When required, NT and the NT receptor antagonist SR48692 were added to the cultures using the following experimental protocol: NT was added 50 min prior to OGD and maintained in contact with cells during the OGD. The NT receptor antagonist SR48692 was added 20 min prior to NT and maintained in contact with cells during NT and OGD exposure and during the 24-h period after OGD. NT and SR48692 alone were also tested.

Endogenous glutamate levels have been quantified using a high-performance liquid chromatography/fluorimetric detection system, including a precolumn derivatization o-phthaldialdehyde reagent and a Chromsep 5 (C18) column. The mobile phase consisted of 0.1 M sodium acetate, 10% methanol, and 2.5% tetrahydrofuran, pH 6.5. The limit of detection for glutamate was 30 fmol per sample.

The effects of the treatments on endogenous extracellular glutamate levels during the third fraction were reported and expressed as percentage changes of basal values, as calculated by the means of the 2 fractions collected prior to treatment.

Lactate Dehydrogenase Levels

The neuronal death was quantitatively evaluated measuring the lactate dehydrogenase (LDH) levels in the extracellular fluid 24 h after OGD exposure using the Cytotoxicity Detection Kit LDH (Roche, Basel, Switzerland). It has been previously established that LDH release correlates linearly with the number of damaged neurons after toxic insult. Sensitivity was 0.2–2 × 104 cells per well. Background LDH levels were determined in control cell cultures not exposed to OGD and were subtracted from all experimental values. The LDH level corresponding to complete neuronal death was determined by assaying sister cultures exposed to 1 mM glutamate for 24 h. The LDH values were expressed as percentage of the value found with complete neuronal death (100%). NT and SR48692 effects on OGD-induced increase in LDH levels were evaluated by following the same procedure as described for endogenous glutamate experiments.

MTT Assay

The integrity of mitochondrial enzymes in viable neurons was determined with a colorimetric assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) levels. In live cells, mitochondrial enzymes have the capacity to transform MTT into insoluble formazon. Sensitivity was about 2.5 × 104 cells per well. Twenty-four hours after OGD, cultures were incubated with MTT 5 mg/ml for 4 h at 37 °C. The formazon was dissolved in isopropanol with 1 M HCl and colorimetrically (absorbance at 570 nm) quantified. Neuronal viability corresponded to the value of the optical density read at 570 nm. The results were expressed as the percentage of neuronal viability measured in control cell cultures (100%). NT and SR48692 effects on OGD-induced change in mitochondrial enzymes activity were evaluated by following the same procedure as described for endogenous glutamate experiments.

Nuclear Staining with Hoechst 33258

Twenty-four hours after OGD exposure, cells were fixed in 4% paraformaldehyde, rinsed with phosphate-buffered saline (PBS), and then incubated for 20 min at room temperature with Hoechst 33258 (1 μg/ml in PBS). After rinsing with PBS, coverslips were mounted on slides with a solution containing 50% glycerol in 0.044 M citrate, 0.111 M phosphate buffer, pH 5.5, and visualized under a fluorescence microscope. The percentage of cells showing chromatin condensation (fragmented nuclei) was quantified by counting ≥3000 cells under each experimental condition (5 randomly selected fields per well, 9–18 wells per condition per experiment, and 5 independent experiments). NT and SR48692 effects on OGD-induced increase of apoptotic nuclei were evaluated by following the same procedure as described for endogenous glutamate experiments.

Annexin V Staining

Annexin V staining was carried out using human annexin V–fluorescein isothiocyanate (FITC) kit (Bender MedSystems, Burlingame, CA) according to the manufacturer's instructions. Annexin V is a phospholipid-binding protein with high affinity for phosphatidyl serine. Annexin V staining was used to label phosphatidyl serine translocated to the outer membrane surface of several cell types undergoing apoptosis. Cells cultured on coverslips were washed twice with a buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 and then incubated with a solution of 5 μl annexin V–FITC and propidium iodide (1 μg/ml). Stained cells were examined at the fluorescence microscope. To quantify the number of apoptotic cells, the annexin-positive/propidium iodide–negative [AN(+)/PI(−)] immunoreactive cells were counted, and the data were expressed as percent of counted cells. NT and SR48692 effects on OGD-induced increase of annexin V staining were evaluated by following the same procedure as described for endogenous glutamate experiments.

Caspase-3 Activity

Caspase-3 activity was measured in lysates of cortical neurons using the caspACE colorimetric assay system (Promega, Madison, WI) following the instructions of manufacturer. The colorimetric substrate N-acetyl-Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (Ac-DEVD-pNA) provided in the caspACE assay system is labeled with the chromophore p-nitroaniline that is released from the substrate upon cleavage by caspase-3. Free pNA produces a yellow color that is monitored by a spectrophotometer at 405 nm. Briefly, neurons were lysed in ice-cold lysis buffer for 20 min. After removal of cellular debris by centrifugation (10 min at 9000 rpm at 4 °C), the supernatants were used to detect caspase-3 proteolytic activity. Samples were incubated with 200 μM caspase-3 substrate Ac-DEVD-pNA at 37 °C for 4 h and then analyzed at 405 nm in a microtiter plate reader. The protein levels in the lysates were measured with BCA protein assay kit (Pierce Biotechnology, Rockford, IL). NT and SR48692 effects on OGD-induced changes in caspsase-3 activity were evaluated by following the same procedure as described for endogenous glutamate experiments.

MAP2 Immunoreactivity

The neuronal damage was also performed by neurons immunochemical numeration through MAP2 immunocytochemistry. On DIV 8, cells were rinsed in 0.1 M PBS and then fixed using 4% paraformaldehyde in Sorensen's buffer 0.1 M, pH 7.4, for 20 min. After rinses in PBS (3 times for 5 min each), the cells were incubated overnight at 4 °C with the primary antibody rabbit anti-MAP2. Anti-MAP2 antibody was diluted 1:1000 in PBS containing 0.3% Triton X-100 (v/v). The cells were then washed 3 times with PBS and incubated with rhodamine-conjugated antirabbit antibody (Chemicon, Temecula, CA) diluted 1:100 in PBS containing 0.3% Triton X-100 for 60 min at room temperature. After 3 washes in PBS, the cells were mounted in glycerol and PBS (3:1, v/v) containing 0.1% 1,4-phenylenediamine and examined using a Nikon Microphot FXA microscope. Investigation of MAP2 aggregations in dendrites was performed with the ×100 magnification objective and on 30 randomly chosen fields in each coverslip. Subsequently, the number of aggregations was counted and referred to 100 μm of dendrite length (Image-Pro Plus 4.1; Immagini e Computer, Milan, Italy) (Pirondi et al. 2005). NT and SR48692 effects on OGD-induced increase of MAP2 immunoreactivity were evaluated by following the same procedure as described for endogenous glutamate experiments.

Materials

The culture dishes were purchased from Nunc A/S (Roskilde, Denmark). Neurobasal medium and B27 were obtained from Gibco (Grand Island, NY). Poly-L-lysine, trypsin, cytosine arabinoside, gentamycine sulfate, glutamine, L-glutamic acid, MTT, and Hoechst 33258 were obtained from Sigma Chemical Co., St Louis, Missouri. Anti-MAP2 antibody and rhodamine-conjugated antirabbit antibody were purchased from Chemicon. NT from Peninsula Laboratories Europe Ltd (Merseyside, UK) was dissolved in Krebs solution just before testing and used only once. SR48692 (2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy-phenyl)pyrazol-3-yl) carboxylamino]tri-cyclo(3.3.1.1.3.7)-decan-2-carboxylic acid) (Sanofi-Aventis, Montpellier, France) was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was < 0.001%; when required, the DMSO vehicle was added alone or together with NT, and no changes in glutamate efflux, LDH levels, mitochondrial enzymes activity, chromatin condensation, caspase-3 activity, annexin V staining, and MAP2 immunoreactivity were observed.

Statistical Analysis

Results are expressed throughout as means ± standard error of mean. The statistical analysis was carried out by analysis of variance followed by the Newman–Keuls test for multiple comparisons. P < 0.05 was the accepted level of significance.

Results

Endogenous Extracellular Glutamate Levels

Modulation of Endogenous Extracellular Glutamate Levels in Cortical Cell Cultures Exposed to OGD

The mean basal extracellular glutamate levels measured in the first 2 samples collected from cell cultures were 0.384 ± 0.021 μM (n = 35). As shown in Figure 1, endogenous extracellular glutamate levels, measured 24 h after OGD, were significantly increased (437 ± 35% of the respective basal value, n = 15) compared with control group not exposed to OGD (293 ± 21% of the respective basal value, n = 10).

Figure 1.

Effects of NT, SR48692 (SR), and NT + SR on endogenous extracellular glutamate levels in cortical cell cultures not exposed and exposed to OGD. Two samples, collected every 50 min, have been used to assess basal glutamate levels. A third fraction was collected 24 h later. The effects of the treatments on the endogenous extracellular glutamate levels in the third fraction are reported and expressed as percent changes of basal values, as calculated by the means of the 2 fractions collected prior to treatment. *P < 0.05 and **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °P < 0.05 and °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 1.

Effects of NT, SR48692 (SR), and NT + SR on endogenous extracellular glutamate levels in cortical cell cultures not exposed and exposed to OGD. Two samples, collected every 50 min, have been used to assess basal glutamate levels. A third fraction was collected 24 h later. The effects of the treatments on the endogenous extracellular glutamate levels in the third fraction are reported and expressed as percent changes of basal values, as calculated by the means of the 2 fractions collected prior to treatment. *P < 0.05 and **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °P < 0.05 and °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Receptor Antagonist SR48692 Alone or in Combination

The addition of NT (100 nM) to the cell cultures exposed to OGD was associated with a supplementary enhancement of the OGD-induced increase of endogenous extracellular glutamate levels (540 ± 47% of the respective basal value, n = 14). The exposure of cells to the NT antagonist SR48692 (100 nM) prevented the effect of the peptide on the OGD-induced increase of glutamate efflux (270 ± 28% of the respective basal value, n = 14). Interestingly, SR48692 100 nM by itself reduced the OGD-induced increase of endogenous extracellular glutamate levels (332 ± 37% of the respective basal value, n = 14) (Fig. 1).

As shown in Figure1, when NT and SR48692 were added, alone or in combination, to cell cultures not exposed to OGD, they did not significantly influence the endogenous extracellular glutamate levels (292 ± 19%, n = 15; 340 ± 26%, n = 10; 336 ± 38%, n = 10, respectively) as compared with the control cell cultures.

Neuronal Death

LDH Release

The LDH level corresponding to complete neuronal death was determined by assaying sister cultures exposed to 1 mM glutamate for 24 h. The LDH values were expressed as percentage of the value found with complete neuronal death (100%).

The LDH release/absorbance, measured in cortical cell cultures not exposed to OGD, was 0.722 ± 0.04 (n = 28) and corresponds to 37 ± 2.8% of complete neuronal death. The exposure of cortical cell cultures to OGD induced a significant increase of LDH release (66 ± 2.6% of complete neuronal death, n = 35) with respect to control cell cultures not exposed to OGD (Fig. 2).

Figure 2.

Effects of NT, SR48692 (SR), and NT + SR on LDH release in cortical cell cultures not exposed and exposed to OGD. The LDH values are expressed as percentage of the value found with complete neuronal death (100%), determined by assaying sister cultures exposed to 1 mM glutamate for 24 h. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 2.

Effects of NT, SR48692 (SR), and NT + SR on LDH release in cortical cell cultures not exposed and exposed to OGD. The LDH values are expressed as percentage of the value found with complete neuronal death (100%), determined by assaying sister cultures exposed to 1 mM glutamate for 24 h. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Receptor Antagonist SR48692 Alone or in Combination

The addition of NT (100 nM) to the cultures further enhanced the OGD-induced increase of LDH release (82 ± 2.8% of complete neuronal death, n = 23). The selective NT receptor antagonist SR48692 (100 nM) prevented not only the effect of the peptide but also the OGD-induced increase of LDH release (47 ± 2.5% of complete neuronal death, n = 25, and 49 ± 2.5% of complete neuronal death, n = 28, respectively; Fig. 2).

Any effects were observed after the addition of NT and SR48692, alone or in combination, to the cultures not exposed to OGD (40 ± 4% of complete neuronal death, n = 22; 43 ± 3% of complete neuronal death, n = 23; 43 ± 4% of complete neuronal death, n = 21, respectively; Fig. 2).

MTT Assay

The MTT absorbance values, measured in cortical cell cultures not exposed to OGD, were 0.125 ± 0.02, n = 20.

Exposure of cortical cell cultures to OGD induced an impairment of oxidative ability of mitochondria, as indicated by MTT reduction, as measured after the insult (68 ± 4% of control value, n = 20; Fig. 3).

Figure 3.

Effects of NT, SR48692 (SR), and NT + SR on neuronal viability (expressed as MTT reduction) in cortical cell cultures not exposed and exposed to OGD. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 3.

Effects of NT, SR48692 (SR), and NT + SR on neuronal viability (expressed as MTT reduction) in cortical cell cultures not exposed and exposed to OGD. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Receptor Antagonist SR48692 Alone or in Combination

The addition of NT (100 nM) to the cultures further decreased the OGD-induced MTT reduction (55 ± 2% of control value, n = 20). SR48692 (100 nM) prevented not only the effect of the peptide but also the OGD-induced MTT reduction (93 ± 4% of control value, n = 18; 91 ± 4% of control value, n = 18, respectively; Fig. 3).

Any effects were observed after the addition of NT and SR48692, alone or in combination, to the cultures not exposed to OGD (92 ± 6% of control value, n = 10; 93 ± 6% of control value, n = 12; 95 ± 5% of control value, n = 12, respectively; Fig. 3).

Nuclear Staining with Hoechst 33258

The specific DNA stain Hoechst 33258 was used to assess changes in chromatin and nuclear structure. As reported in a representative fluorescence photomicrograph (Fig. 4A), nuclei of viable cells (control group, not exposed to OGD) exhibited a large and diffuse chromatin staining. In contrast, nuclei of cortical cells exposed to OGD showed a variety of abnormal morphologies including highly condensed and fragmented chromatin (Fig. 4B). All nuclei with morphological abnormalities were considered “condensed.” In OGD-exposed cell cultures, the percentage of cells with altered nuclear morphology (fragmented nuclei) was significantly higher than that observed in control cell cultures not exposed to OGD (55 ± 3.8%, n = 18, and 32 ± 2.3%, n =15, respectively; Fig. 5).

Figure 4.

Representative fluorescence photomicrographs of cells with nuclear fragmentation in cortical cell cultures not exposed (panel A) and exposed to OGD (panel B), to OGD + NT (panel C), and to OGD + NT + SR48692 (panel D). The neurons were stained with Hoechst 33258 and observed in sampled fields under fluorescent microscope (magnification ×20).

Figure 4.

Representative fluorescence photomicrographs of cells with nuclear fragmentation in cortical cell cultures not exposed (panel A) and exposed to OGD (panel B), to OGD + NT (panel C), and to OGD + NT + SR48692 (panel D). The neurons were stained with Hoechst 33258 and observed in sampled fields under fluorescent microscope (magnification ×20).

Figure 5.

Effects of NT, SR48692 (SR), and NT + SR on the percentage of fragmented nuclei in cortical cell cultures not exposed and exposed to OGD. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 5.

Effects of NT, SR48692 (SR), and NT + SR on the percentage of fragmented nuclei in cortical cell cultures not exposed and exposed to OGD. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Receptor Antagonist SR48692 Alone and in Combination

The addition of NT (100 nM) to the cultures further enhanced the OGD-induced increase in nuclear fragmentation (Fig. 4C) and in the percentage of fragmented nuclei (71 ± 3%, n = 9; Fig. 5). In the presence of the selective NT receptor antagonist SR48692 (100 nM), not only the NT-induced effect was counteracted (Fig. 4D) but also the OGD-induced increase of the percentage of fragmented nuclei was prevented (41 ± 1.4%, n = 9, and 43 ± 1.5%, n = 8, respectively; Fig. 5).

Any effects were observed after the addition of NT and SR48692, alone or in combination, to the cultures not exposed to OGD (35 ± 4%, n = 8; 33 ± 3%, n = 8; 34 ± 4%, n = 8, respectively; Fig. 5).

Annexin V Staining

AN/PI double staining was used to demonstrate that translocation of phosphatidyl serine from the inner to the outer leaflet, a characteristic feature of apoptotic death, occurs after OGD in cortical cell cultures. The cells stained by annexin V but not by propidium iodide [AN(+)/PI(−)] are those that undergo apoptotic cell death. In normoxic conditions, the cultures showed that 4.5 ± 0.5% (n = 9) of the total cells counted were AN(+)/PI(−), whereas in the OGD-exposed cultures the number of AN(+)/PI(−) immunoreactive cells increased to 9 ± 0.9% (n = 9) (Fig. 6).

Figure 6.

Effects of NT, SR48692 (SR), and NT + SR on the percentage of AN(+)/PI(−) immunoreactive cells in cortical cell cultures not exposed and exposed to OGD. *P < 0.05 and **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD+ SR + NT; ΔΔP < 0.01 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 6.

Effects of NT, SR48692 (SR), and NT + SR on the percentage of AN(+)/PI(−) immunoreactive cells in cortical cell cultures not exposed and exposed to OGD. *P < 0.05 and **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD+ SR + NT; ΔΔP < 0.01 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Receptor Antagonist SR48692 Alone and in Combination

The addition of NT (100 nM) to the cultures further enhanced the OGD-induced increase in the percentage of AN(+)/PI(−) immunoreactive cells (13 ± 0.8%, n = 9; Fig. 6). In the presence of the selective NT receptor antagonist SR48692 (100 nM), the NT-induced effect was counteracted (9 ± 1%, n = 8; Fig. 6).

Any effects were observed after the addition of NT and SR48692, alone or in combination, to the cultures not exposed to OGD (5 ± 1%, n = 8; 4 ± 0.3%, n = 8; 4.5 ± 0.5%, n = 8, respectively; Fig. 6).

Caspase-3 Activity

In cortical cell cultures not exposed to OGD, the caspase-3 activity was 6.9 ± 0.6 (pNA pmol/min/mg protein; n = 10). Exposure of cortical cell cultures to OGD induced an increase of caspase-3 activity (135 ± 10% of control value, n = 10; Fig. 7).

Figure 7.

Effects of NT, SR48692 (SR), and NT + SR on the caspase-3 activity expressed as percentage of control values in cortical cell cultures not exposed and exposed to OGD. *P < 0.05 and **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °P < 0.05 significantly different from OGD + SR and OGD+ SR + NT; ΔΔP < 0.01 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 7.

Effects of NT, SR48692 (SR), and NT + SR on the caspase-3 activity expressed as percentage of control values in cortical cell cultures not exposed and exposed to OGD. *P < 0.05 and **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °P < 0.05 significantly different from OGD + SR and OGD+ SR + NT; ΔΔP < 0.01 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Receptor Antagonist SR48692 Alone or in Combination

The addition of NT (100 nM) to the cultures further enhanced the OGD-induced increase of caspase-3 activity (206 ± 18% of control value, n = 10; Fig. 7). In the presence of SR48692 (100 nM), the NT-induced increase of caspase-3 activity was counteracted (155 ± 16% of control value, n = 10; Fig. 7).

Any effects were observed after the addition of NT and SR48692, alone or in combination, to the cultures not exposed to OGD (102 ± 9% of control value, n = 10; 103 ± 9.5% of control value, n = 12; 98 ± 8% of control value, n = 10, respectively; Fig. 7).

MAP2 Immunoreactivity

As MAP2 is described as an early indicator of ischemia-induced neurodegeneration, the cortical cell cultures were MAP2 immunostained. In normoxic conditions, the cultures showed a high number of healthy neurons and a neuronal network formed by highly arborized dendritic trees and an homogeneously diffused MAP2 immunoreactivity of the cell bodies and dendrites (see Fig. 8A,A’). On the contrary, a loss of MAP2 immunoreactivity was observed after OGD in particular in the dendrites, which often appeared to be truncated, as showed by the different distribution of MAP2 immunoreactivity along the dendrites (Fig. 8D,D’).

Figure 8.

Representative fluorescence photomicrographs of MAP2 immunoreactivity in cortical cell cultures not exposed (control: A, A’) and exposed to OGD (D, D’). Effects of NT 100 nM (B, B’) and SR48692 100 nM (C, C’) in cortical cell cultures not exposed to OGD. Effects of NT 100 nM (E, E’), SR48692 100 nM (F, F’), and NT 100 nM + SR48692 100 nM (G, G’) in cortical cell cultures exposed to OGD. Surviving neurons were stained with anti-MAP2 antibody and observed in sampled fields under fluorescent microscope 24 h after OGD (magnification ×20: A, B, C, D, E, F, G; magnification ×40: A’, B’, C’, D’, E’, F’, G’).

Figure 8.

Representative fluorescence photomicrographs of MAP2 immunoreactivity in cortical cell cultures not exposed (control: A, A’) and exposed to OGD (D, D’). Effects of NT 100 nM (B, B’) and SR48692 100 nM (C, C’) in cortical cell cultures not exposed to OGD. Effects of NT 100 nM (E, E’), SR48692 100 nM (F, F’), and NT 100 nM + SR48692 100 nM (G, G’) in cortical cell cultures exposed to OGD. Surviving neurons were stained with anti-MAP2 antibody and observed in sampled fields under fluorescent microscope 24 h after OGD (magnification ×20: A, B, C, D, E, F, G; magnification ×40: A’, B’, C’, D’, E’, F’, G’).

To quantify the effect of OGD on cell morphology, the number of MAP2 aggregations in dendrites was counted and referred to 100 μm of dendrite length sections. As shown in Figure 9, in control cultures not exposed to OGD, MAP2 aggregations per length unit were 1.24 ± 0.98 aggregation per 100 μm (n = 10), whereas cultures exposed to OGD showed a marked increase (12.30 ± 1.10 aggregation/100 μm, n = 10).

Figure 9.

Effects of NT, SR48692 (SR), and NT + SR on the number of MAP2 aggregations per 100 μm length in cortical cell cultures not exposed and exposed to OGD. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Figure 9.

Effects of NT, SR48692 (SR), and NT + SR on the number of MAP2 aggregations per 100 μm length in cortical cell cultures not exposed and exposed to OGD. **P < 0.01 significantly different from control as well as NT, SR, and NT + SR (not exposed to OGD); °°P < 0.01 significantly different from OGD + SR and OGD + SR + NT; ΔP < 0.05 significantly different from OGD according to analysis of variance followed by the Newman–Keuls test for multiple comparisons.

Effects of the Exposure to NT and NT Antagonist SR48692 Alone and in Combination

When NT (100 nM) was added to the OGD-exposed cultures, the fragmented distribution of MAP2 immunostaining along the dendrites became more noticeable (Fig. 8E,E’), and a further significant increase in MAP2 aggregations was observed (17.55 ± 1.21 aggregation per 100 μm; Fig. 9). The addition of SR48692 (100 nM) not only counteracted the NT-induced effect but also the OGD-induced decrease of dendrite outgrowth (Fig. 8F,F’, respectively) and OGD-induced increase of MAP2 aggregations (1.87 ± 0.69 aggregation per 100 μm, n = 11; 2.12 ± 0.95 aggregation per 100 μm, n = 11, respectively; Fig. 9).

The addition of NT and SR48692, alone or in combination, to the cultures not exposed to OGD did not modify the dendrite outgrowth (Fig. 8B,B’,C,C’, respectively) and the number of MAP2 aggregations (1.30 ± 0.75 aggregation per 100 μm, n = 8; 0.53 ± 0.09 aggregation per 100 μm, n = 8; 1.39 ± 0.85 aggregation per 100 μm, n = 8, respectively; Fig. 9).

Discussion

The OGD is an in vitro model of ischemia (Strasser and Fischer 1995) that has been used in this study to define the role of NT in OGD-induced neuronal death in cortical cell cultures and also to give a pathological correlate to the ability of NT to enhance NMDA-induced glutamate release (Antonelli et al. 2004). Furthermore, glucose insufficiency reduces neuronal viability and increases caspase-3 activity linked to increased LDH levels (Ioudina et al. 2004). Both necrotic and apoptotic neuronal death have been described following cerebral ischemia.

The use of diverse methods for measuring cell death should help to define causal relationship between the mechanisms that regulate apoptosis and the cell death event itself. Biochemical methods such as LDH release and MTT enzyme activity, used to quantify the cell death, analyze different cellular events. Indeed, LDH release serves as an indicator of loss of cell membrane integrity and thus cell death. The MTT assay provides an indirect measurement of cell growth/cell death through measurement of the ability of mitochondria to convert MTT to formazon. This assay measures mitochondrial function and not cell death per se. The measurement of loss of mitochondrial function will be a late indicator of apoptotic cell death because mitochondria often remain intact until late in apoptosis. These biochemical methods present several drawbacks; indeed, both LDH release and MTT assay do not discriminate between apoptosis and necrosis. Thus, different methods are required to confirm apoptosis (Loo and Rillema 1998). Apoptosis is characterized by nuclear changes, that is, aggregation of chromatin at the nuclear membrane, membrane blebbing without loss of integrity, and chromatin fragmentation (Simm et al. 1997). For this reason, the specific DNA stain, Hoechst 33258, was used to assess changes in chromatin and nuclear structure following OGD. Furthermore, caspase-3 activity and annexin V assay were used to confirm the presence of the apoptotic component in OGD-induced neuronal cell death. Annexin V assay is based on the observation that during induction of apoptosis, phosphatidyl serine (PS) is translocated from the inner leaflet to the outer leaflet of the plasma membrane (Huerta et al. 2007). Under the OGD condition, cultured neurons exhibited annexin V labeling on the periphery of their soma and dendrites while PI was excluded. These data confirm that PS is externalized, whereas the plasma membrane remains intact, strongly supporting the occurrence of apoptosis. Finally, the MAP2 family of proteins is an abundant group of cytoskeletal components that are predominantly expressed in neurons and have been proposed to play important roles in the outgrowth of neuronal processes, synaptic plasticity, and neuronal cell death. MAP2 is depleted after in vivo and in vitro ischemia (Li et al. 2000; Kuhn et al. 2005), and for this reason, MAP2 immunoreactivity may be considered an early indicator of ischemia-induced neurodegeneration. (Buddle et al. 2003). Thus, by measuring extracellular glutamate levels, LDH levels, mitochondrial dehydrogenase activity, apoptotic nerve cell death with Hoechst 33258, annexin V and caspase-3 activity, and MAP2 immunoreactivity, we provide biochemical and morphological evidence that NT, via the activation of NTR1, is involved in causing neuronal death upon OGD in cortical cultures. In fact, the addition to the incubation medium of exogenous NT at nanomolar concentrations during OGD produces a further and significant enhancement of the LDH levels and a further significant decrease of mitochondrial functionality with respect to the LDH and MTT levels observed during OGD alone. In line with the above finding, it has been shown that the NTR1 antagonist SR48692 can increase cell membrane integrity and cell viability.

The possible role of the NTR1 in degeneration induced by OGD is further supported by the results obtained in the experiments where the apoptotic cell death is analyzed. In fact, these data indicate that the peptide on its own enhances, whereas the NT receptor antagonist blocks the OGD-induced increase in the number of fragmented nuclei and AN(+)/PI(−) immunoreactive cells as well as caspase-3 activity in the cortical cultures. In addition, the outgrowth of cortical dendrites, already altered after OGD (Matesic and Lin 1994; Vanicky et al. 1995), are further impaired in the presence of NT, as demonstrated by the MAP2 immunoreactivity experiments. The injury to the neuronal dendrites, which may be observed before neuronal death, is characterized by a reduction of outgrowth and branching of dendrites as well as by an increase of the number of MAP2 aggregations. This alteration in dendrite morphology is an early consequence of excessive glutamate release occurring during ischemia (Esquenazi et al. 2002). The involvement of NTR1 in the OGD-induced reduction of neuronal population and dendritic outgrowth is confirmed by the treatment with SR48692, which antagonizes the OGD-induced neurodegeneration.

Thus, taken all together these biochemical and morphological results lead to hypothesize that under normothermic conditions NTR1 activation may contribute to apoptotic nerve cell death induced by OGD with reduction of neuronal survival (see Ioudina et al. 2004).

Among the myriad of biochemical events triggered by cerebral ischemia, increase in Ca2+ influx, formation of free radicals, loss of adenosine triphosphate, etc., there is also an increase in extracellular concentration of neurotransmitters, especially of glutamate. In this context, it is well known that substantial elevation in extracellular glutamate levels and, consequently, the excessive stimulation of excitatory aminoacid receptors is implicated in the neuronal cell death occurring under these degenerative processes (Choi et al. 1988; Arundine and Tymianski 2004; Young et al. 2004; Zipfel et al. 2000). Thus, in view of the results obtained in the present study, it could be suggested that one of the possible mechanisms that leads to NT-mediated apoptotic nerve cell death and NT-mediated reduction of dendritic outgrowth and branching could involve the ability of the peptide to modulate the glutamatergic transmission.

Such a hypothetical mechanism seems to be confirmed by the results indicating that during the OGD, the presence of NT in the medium induces a significant amplification in the rise of extracellular glutamate levels compared with that observed during OGD alone. This view is strengthened by the result indicating that the NT receptor antagonist SR48692 counteracts the OGD-induced rise of the extracellular levels of glutamate reducing the endogenous levels of glutamate to the values observed in control neuronal cells. Interestingly, SR48692 is able to counteract some biochemical and morphological OGD-induced alterations also in the absence of exogenous NT. These findings can be explained by release of endogenous NT from neuronal and/or glial cells under OGD increasing NTR1 tone that can be blocked by the NTR1 receptor antagonist. In line with this explanation, a rise of NT immunoreactivity in certain brain regions has been found in the rat middle cerebral artery occlusion model of stroke (Allen et al. 1995). However, SR48692 by itself did not reduce the caspase-3 activation and the number of AN(+)/PI(−) immunoreactive cells induced by OGD. At the present, this discrepancy could be attributed to the different methodological approaches used to determine neurodegeneration and remain to be clarified in further studies.

It is worth noting that previous results performed in mesencephalic cell cultures containing dopaminergic neurons that express functional NT receptors demonstrate that the neurotoxic effects of glutamate are exacerbated by NT when the peptide is applied in combination with exogenous glutamate (Antonelli et al. 2002). NTR1 is coupled to phospholipase C and will therefore upon activation increase protein kinase C activity (see Kinkead and Nemeroff 2004), and it has previously been demonstrated that NT receptors enhance NMDA-induced excitotoxicity in cortical cultures via an activation of protein kinase C (Antonelli et al. 2004). Therefore, it may be speculated that under the present pathological conditions, the peptide both by enhancing glutamate release and the NT–NMDA receptor interactions could amplify the glutamate signal transduction contributing to neuronal injury after both OGD and probably after ischemia. Thus, it is known that protein kinase C when activated can increase the opening rate of the NMDA channels and recruit new NMDA channels to the surface membrane (see Lan et al. 2001). This amplification of NMDA signaling by NT could explain the increase of MAP2 aggregations occurring after OGD. In fact, following NMDA receptor activation, the calcium influx, through the receptor ion channel complex, and the release of calcium from the mitochondria, through the activation of the 2Na+/Ca++ exchanger, trigger MAP2 degradation (Buddle et al. 2003).

Hypothermia affects a wide variety of processes involved in the cerebral ischemia, and in both models of global and focal cerebral ischemia, it has been shown that hypothermia could be neuroprotective (Corbett and Thornhill 2000; Gordon 2001; Krieger and Yenari 2004). Thus, an alternative to the classical pharmacological approach to obtain neuroprotection could be the administration of a compound that induces hypothermia. In this context, it has been suggested that the hypothermic effect of NT could be the mechanism involved in the reduction of the CA1 damage in hippocampus after global ischemia in mongolian gerbils (Babcock et al. 1993). In addition, NT and NT analogues have been reported to produce hypothermia in rodents (Bissette et al. 1976; Dubuc et al. 1992), and the neuroprotective effect of the NT analogue H-Lys-psi (CH2NH)Lys-Pro-Tyr-Ile-Leu-OH (JMV-449) has been demonstrated in a mouse model of permanent distal middle cerebral artery occlusion (Torup et al. 2003). Such a neuroprotection is likely to be mediated via the systemic hypothermia as no neuroprotective effect was seen if the JMV-449–treated mice were kept normothermic (Torup et al. 2003). However, it must be noted as shown in the present study that the NTR1 antagonist SR48692 in vitro counteracts the local neurodegeneration-enhancing effects of NT, whereas the hypothermic effects of NT in vivo are not counteracted by this NTR1 antagonist (Gully et al. 1995, 1997). Thus, NTR1 antagonist such as SR48692 may offer a new treatment possibility of ischemia.

In conclusion, taken together the present results, obtained in the OGD model in cortical cultures representing an in vitro model of cortical ischemia, suggest that cortical NT receptor activation may contribute to neuronal injury after ischemia. The NT receptor antagonists could provide a new tool to explore the clinical possibilities and thus to move from chemical compound to effective drug.

Funding

Sanofi-Aventis, Toulose, France.

The authors thank Fondazione Cassa di Risparmio di Ferrara, Italy, and IRET-Foundation, Italy. Conflict of Interest: None declared.

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