Neuronal Death Enhanced by N-Methyl-D- Aspartate Antagonists

Neuronal death enhanced by N-methyl-D- aspartate antagonists

Chrysanthy Ikonomidou*†, Vanya Stefovska*, and Lechoslaw Turski‡

*Department of Pediatric Neurology, Children’s Hospital, Charite´-Virchow Clinics, Humboldt University, Augustenburger Platz 1, D-13353 Berlin, Germany; and ‡Eisai London Research Laboratories, Bernard Katz Building, University College London, Gower Street, London, WC1E 6BT, United Kingdom

Communicated by Martin Lindauer, University of Wu¨rzburg, Wu¨rzburg, Germany, August 28, 2000 (received for review May 23, 2000) Glutamate promotes neuronal survival during brain development thesia. 3NP was infused at 12 or 24 mg͞kg per d for 28 days. and destroys neurons after injuries in the mature brain. Glutamate Dizocilpine (MK801; 0.3 mg͞kg per d), memantine (24 mg͞kg antagonists are in human clinical trials aiming to demonstrate per d), 3-((Ϯ)-2-carboxypiperazin-4-yl)propyl-1-phosphonate limitation of neuronal injury after head trauma, which consists of (CPP; 24 mg͞kg per d), 2,3-dihydroxy-6-nitro-7-sulfamoylben- both rapid and slowly progressing neurodegeneration. Further- zo(f)quinoxaline (NBQX; 24 mg͞kg per d), [1,2,3,4-tetrahydro- more, glutamate antagonists are considered for neuroprotection in 7-morpholinyl-2,3-dioxo-6-(trifluoromethyl)quinoxalin-1- chronic neurodegenerative disorders with slowly progressing cell yl]methylphosphonate (MPQX; 24 mg͞kg per d), or vehicle were death only. Therefore, humans suffering from Huntington’s dis- administered s.c. by means of osmotic minipumps either simul- ease, characterized by slowly progressing neurodegeneration of taneously with 3NP, 12 mg͞kgperdor24mg͞kg per d, or alone. the basal ganglia, are subjected to trials with glutamate antago- Intrastriatal microinjections. Wistar rats were anesthetized with nists. Here we demonstrate that progressive neurodegeneration sodium pentobarbital (50 mg͞kg i.p.) and subjected to bilateral in the basal ganglia induced by the mitochondrial toxin 3-nitro- microinjection of 3NP, 100, 250, or 500 nmol, into the striatum propionate or in the hippocampus by traumatic brain injury is at coordinates derived from the stereotaxic atlas of Swanson enhanced by N-methyl-D-aspartate antagonists but ameliorated by (10). The coordinates were: AP (anterior͞posterior) 7.63; L ␣-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonists. (lateral) 2.0; V (ventral) 3.2. To assess effect of NMDA antag- These observations reveal that N-methyl-D-aspartate antagonists may increase neurodestruction in mature brain undergoing slowly onists on neurodegeneration induced by 3NP, CPP, 250 nmol, progressing neurodegeneration, whereas blockade of the action of was coadministered with 3NP, 250 nmol, into one striatum. The ␣ contralateral side received 3NP alone and served as control. glutamate at -amino-3-hydroxy-5-methyl-4-isoxazole-propionate ␮ receptors may be neuroprotective. Drugs were delivered into the striatum in a volume of 2 lata rate of 0.1 ␮l͞min. Neurologic assessment. The following scoring system was used lutamate antagonists were demonstrated to be neuropro- to grade neurologic impairment in rats subjected to treatment Gtective in stroke and head trauma in rodents and nonhuman with 3NP: 0, no observable motor deficits; 1, reduction of primates (1, 2). Accordingly, the excitatory neurotransmitter spontaneous locomotor activity; 2, unsteady, wobbly gait, ataxia; glutamate has been pathogenetically linked to cell death in acute and 3, severe depression of movement and loss of righting reflex. neurodegenerative disorders in humans such as stroke or trau- Scores were taken three times a week by a single observer under matic brain injury (1). This inference prompted the assumption blinded conditions. Shown are mean Ϯ SEM maximal scores that glutamate antagonists must be neuroprotective in chronic neurodegenerative disorders in humans (3, 4). However, whether registered during the entire observation period. Morphology. glutamate antagonists limit neurodegeneration in slowly pro- For morphological examination, rats were anes- gressing neurodegenerative disorders is not known. Neverthe- thetized with an overdose of pentobarbital and perfused with a less, clinical trials in humans suffering from Huntington’s dis- fixative containing 1% paraformaldehyde and 1.5% glutaralde- ease, Parkinson’s disease, and severe dementia using glutamate hyde in pyrophosphate buffer (for combined light and electron N-methyl-D-aspartate (NMDA) antagonists are in progress (5). microscopy), or containing 10% acetic acid, 10% formaldehyde, Neuronal loss in the basal ganglia is the hallmark pathological 80% methanol (for paraffin embedding). Serial coronal sections ␮ feature of Huntington’s disease and can be reproduced in rodents of the whole brain were cut 10 m thick, and every 20th section and primates by administration of the succinate dehydrogenase was mounted on a glass slide and stained with cresyl violet, or by inhibitor 3-nitropropionate (3NP) (6). Intrastriatal administration Fink and Heimer technique (11). For electron microscopy of 3NP produces in rodents rapid neuronal death and dystonia striatal tissue was processed in osmium tetroxide, dehydrated in resembling that seen in humans suffering from moldy sugarcane graded ethanols, cleared in toluene, embedded in araldite, and poisoning (7). Systemic administration of 3NP produces slowly examined by transmission electron microscope. progressing neuronal death in the striatum of rodents and primates Quantiﬁcation of neuronal damage in the striatum. The volume with symptoms of chorea developing with a considerable delay (8). of the striatum in rats subjected to systemic treatment with 3NP, Similarly, traumatic head injury causes immediate death of neurons glutamate antagonists, or vehicle was measured 3 days after at impact in the cortex and slowly progressing neurodegeneration termination of continuous administration of drugs, by using an at sites distant to the impact such as hippocampus (9). image analysis system. To provide an estimate for the overall Here we demonstrate that slowly progressing neuronal death induced by systemic treatment with 3NP in the striatum or by traumatic brain injury in the hippocampus is enhanced by Abbreviations: AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazole-propionate; CPP, 3-((Ϯ)-2- NMDA but ameliorated by ␣-amino-3-hydroxy-5-methyl-4- carboxypiperazin-4-yl)propyl-1-phosphonate; MK801, dizocilpine; MPQX, [1,2,3,4- tetrahydro-7-morpholinyl-2,3-dioxo-6-(triﬂuoromethyl)quinoxalin-1-yl]methylphospho- isoxazole-propionate (AMPA) antagonists in mature rodent nate; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline; NMDA, N-methyl-D- brain. Rapidly progressing neuronal death induced by intrastri- aspartate; 3NP, 3-nitropropionate; Nv, numerical density. atal treatment with 3NP or by traumatic brain injury in the cortex †To whom reprint requests should be addressed. E-mail: [email protected]. is decreased by NMDA antagonists. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Materials and Methods §1734 solely to indicate this fact.

3NP. Systemic delivery. Wistar rats (24–28 months old, 750–950 g) Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.220412197. PHARMACOLOGY were implanted s.c. with osmotic minipumps under ether anes- Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.220412197

PNAS ͉ November 7, 2000 ͉ vol. 97 ͉ no. 23 ͉ 12885–12890 Downloaded by guest on September 28, 2021 striatal neuronal loss over the treatment period of 28 days, administration of vehicle, CPP, or NBQX. The pH, arterial numerical densities (Nv) of striatal neurons were determined by oxygen, carbon dioxide, glucose, and bicarbonate levels were means of the stereologic disector (12, 13) and the total number determined by using automated diagnostic procedures. A rectal of neurons remaining in the striatum were calculated. The temperature probe was connected to a temperature controller volume of the striatum and striatal damage resulting from coupled to a heating pad maintained at 37.5 Ϯ 0.5°C throughout intrastriatal microinjections of 3NP was estimated volumetrically surgery. After surgery, animals were transferred to individual 4 h after administration by using image analysis. To provide an home cages that were kept on heated tables set at 36.5–37.5°C for estimate for neuronal loss after microinjection of 3NP, CPP, or up to 24 h. During the following 48 h animals were individually vehicle into the striatum, Nv of normal neurons were determined housed under standard environmentally controlled conditions (6 in striatum by means of the stereologic disector and the numbers a.m. to 6 p.m.; 12-hr light͞dark cycle; 24–25°C) and were of neurons lost in the striatum were calculated. permitted free access to food and water. Body weight was monitored by means of a Sartorius model U6100 balance. Traumatic Brain Injury. Contusing device. The contusing device Morphometric analysis in cortex and hippocampus. The volume consisted of a stainless steel tube, 40 cm in length, perforated at of the damage in the cortex was determined stereologically 3 1-cm intervals to prevent air compression in the tube. Fischer 344 days after traumatic injury by using an image analysis. The rats, 230–270 g, were anesthetized with tribromoethanol, 260 damage in the hippocampal CA3 subfield was determined mg͞kg i.p. A craniotomy over the right hemisphere was made, stereologically at 13 different rostrocaudal levels extending from the device guiding a falling weight onto the footplate resting on 9.67 to 12.21 mm (10) and throughout its mediolateral axis 3 days the surface of the dura was placed perpendicular to the surface after traumatic injury. To quantitatively assess neuronal loss in of the skull, and a force of 380 g ϫ cm produced by a 20-g weight the hippocampus, stereological disector technique was used to was selected to produce brain contusion. A maximum of 2.5 mm estimate Nv of pyramidal neurons, and the numbers of neurons depression of the brain surface was allowed to avoid mechanical remaining in the CA3 subfield were calculated. An unbiased puncture of the dura. The center of the footplate was stereotaxi- counting frame (0.05 mm ϫ 0.05 mm; disector height 0.01 mm) cally positioned 1.5 mm posterior and 2.5 mm lateral to the and a high-aperture objective (ϫ100) were used for sampling. bregma. The contusion was made to the cortex corresponding to Normal neurons were identified by the presence of the typical areas Fr1, Fr2, HL, and FL (10). Sham controls were subjected nuclei with clear nucleoplasm and distinct nucleolus surrounded to anesthesia and surgery without the injury. The rats underwent by cytoplasm containing Nissl substance. The border between perfusion fixation 3 days after brain injury. The treatment CA2 and CA3 subfields was considered as the point where the regimen with CPP, three hourly doses of 30 mg͞kg i.p., was looser arrangement of large pyramidal cells goes into densely chosen to assure that relevant concentrations in the brain to packed pyramidal cells of the subfield CA3. An arbitrary line interact with NMDA receptors were present. The AMPA͞ connecting the lateral ends of the dentate granule cell layers was kainate antagonist NBQX was used as a drug reference and was considered a junction between subfields CA3 and CA4. given following the same regimen as that of CPP. Treatment with CPP or NBQX was initiated 2 h before, 1, 4, 7, or 10 h after Statistics. Data were analyzed statistically by means of ANOVA, traumatic cortex injury. CPP and NBQX were administered in a Student’s t test, Mann–Whitney u test, and ␹2 test. volume of 0.5 ml͞100 g of body weight. Physiological monitoring. To monitor arterial blood pressure Results and blood gases, catheters were placed in the femoral and tail Systemic Delivery of 3NP and Glutamate Antagonists. To induce arteries. Samples of arterial blood were collected 5 min before slowly progressing neuronal death in the striatum, 3NP, 12 and 24 and 30 min, 1, 2, and3haftertraumatic brain injury and the last mg͞kg per d, was administered systemically to 24- to 28-month-old

Table 1. Dose-response relationship of neurotoxic action of 3NP in the rat striatum after systemic administration over 28 days and effect of the NMDA antagonists CPP, MK801, and memantine, and the AMPA͞kainate antagonists NBQX and MPQX on 3NP toxicity Treatment, Density of neurons in the striatum, 3 6 3 mg͞kg per d Striatal volume, mm NV; mean ϫ 10 ͞mm Ϯ SEM Neurons, mean Ϯ SEM % n

Vehicle 22.56 Ϯ 0.93 0.141 Ϯ 0.003 3,179,719 Ϯ 148,757 100 13 3NP 12 ϩ vehicle 23.05 Ϯ 0.74 0.116 Ϯ 0.004ϩϩ 2,682,490 Ϯ 144,728ϩ 84 15 3NP 24 ϩ vehicle 23.50 Ϯ 0.61 0.057 Ϯ 0.010ϩϩϩ 1,301,823 Ϯ 198,684ϩϩϩ 41 8 3NP 12 ϩ CPP 24 19.99 Ϯ 0.75** 0.084 Ϯ 0.006*** 1,665,659 Ϯ 112,902*** 52 9 3NP 12 ϩ MK801 0.3 20.33 Ϯ 0.91* 0.088 Ϯ 0.006*** 1,766,705 Ϯ 114,380*** 56 6 3NP 12 ϩ Memantine 24 20.42 Ϯ 0.82* 0.086 Ϯ 0.007*** 1,756,120 Ϯ 115,768*** 55 9 Vehicle ϩ CPP 24 22.35 Ϯ 0.32 0.141 Ϯ 0.005 3,156,938 Ϯ 127,962 99 4 Vehicle ϩ MK801 0.3 22.50 Ϯ 0.63 0.141 Ϯ 0.003 3,176,258 Ϯ 99,183 100 6 Vehicle ϩ Memantine 24 22.41 Ϯ 0.41 0.140 Ϯ 0.006 3,137,406 Ϯ 135,672 99 4 3NP 12 ϩ NBQX 24 22.61 Ϯ 0.35 0.136 Ϯ 0.004 3,080,253 Ϯ 79,640* 97 6 3NP 24 ϩ NBQX 24 22.95 Ϯ 0.23 0.130 Ϯ 0.009 3,000,500 Ϯ 217,863$$$ 94 7 Vehicle ϩ NBQX 24 22.63 Ϯ 0.74 0.140 Ϯ 0.004 3,175,564 Ϯ 104,348 100 6 3NP 12 ϩ MPQX 24 22.51 Ϯ 0.32 0.135 Ϯ 0.004 3,036,040 Ϯ 60,090* 95 8 3NP 24 ϩ MPQX 24 22.09 Ϯ 0.39 0.121 Ϯ 0.014 2,663,404 Ϯ 277,681$$$ 84 6 Vehicle ϩ MPQX 24 22.40 Ϯ 0.30 0.141 Ϯ 0.006 3,152,380 Ϯ 125,392 99 8

Morphometric analyses revealed that striatal volume and Nv of striatal neurons were signiﬁcantly reduced in rats subjected to parallel treatment with either 3NP, 12 mg͞kg per d and CPP, or 3NP, 12 mg͞kg per d and MK801, or 3NP, 12 mg͞kg per d and memantine over 28 days. The total number of neurons lost in the striatum after combined treatment with 3NP and CPP, or 3NP and MK801, or 3NP and memantine was signiﬁcantly higher than that in the striatum of rats subjected to 3NP and vehicle. NBQX and MPQX attenuated toxicity of 3NP decreasing loss of striatal volume and numerical density, and reducing loss of neurons in the striatum. The dose of either CPP, MK801, memantine, NBQX, or MPQX chosen for long-term treatment was the maximal tolerated dose that did not induce motor disturbances in rats. ϩ, P Ͻ 0.05; ϩϩ, P Ͻ 0.01; ϩϩϩ, P Ͻ 0.001 vs. vehicle-treated rats; *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001 vs. rats treated with 3NP, 12 mg͞kg per d, and vehicle; $$$, P Ͻ 0.001 vs. rats treated with 3NP, 24 mg͞kg per d, and vehicle (Student’s t test).

12886 ͉ www.pnas.org Ikonomidou et al. Downloaded by guest on September 28, 2021 Fig. 1. Electron microphotographs depicting a normal (A) and degenerating striatal neurons (B and C) in rats subjected to systemic treatment with 3NP, 12 mg͞kg per d, over 7 days before death. The neuron in B has a darkened appearance, the cytoplasm displays ﬁne vacuoles and widened cisternae of the endoplasmic reticulum. Cell organelles appear intact as do the cytoplasmic and nuclear membranes. Nuclear chromatin shows no clumps, nucleolus is intact. The neuron in C has a darker appearance and has shrunk down in size. Small vacuoles and prominent cisternae of the endoplasmic reticulum are also present. At this stage nuclear chromatin has started to form clumps that attach to the nuclear membrane that remains intact. Magniﬁcations: ϫ3,600 in A and B and ϫ4,650 in C.

rats by means of osmotic minipumps over 28 days. Neurological rats subjected to simultaneous treatment with 3NP, 12 mg͞kg per scores assessed three times͞week during treatment with 3NP d. Parallel treatment with MK801 or memantine and 3NP revealed that 12 mg͞kg per d caused little motor impairment (score resulted in worsening of neurologic impairment (score 2.80 Ϯ 1.72 Ϯ 0.23, n ϭ 16). Morphometric analysis of the brains 3 days 0.13, n ϭ 10 for 3NP ϩ MK801 and 2.70 Ϯ 0.3, n ϭ 10 for 3NP after termination of the treatment revealed that 3NP, 12 mg͞kg per ϩ memantine vs. 1.72 Ϯ 0.23, n ϭ 16 for 3NP ϩ vehicle; P Ͻ 0.01, d, caused loss of 16% of neurons in the striatum (Table 1). 3NP, 24 Mann–Whitney u test) and increased mortality (90% and 90% mg͞kg per d, produced severe motor disturbances such as unsteady vs. 37.5%; P Ͻ 0.01, ␹2 test) as compared with treatment with gait, ataxia, and increase of muscle tone (score 2.41 Ϯ 0.21, n ϭ 19) 3NP and vehicle. Administration of CPP and 3NP, 12 mg͞kg per as well as mortality. Morphometric analysis of the brains of rats d, also resulted in worsening of neurologic impairment (score subjected to treatment with 24 mg͞kg per d showed loss of 59% of 2.25 Ϯ 0.16, n ϭ 12; P Ͻ 0.05, Mann–Whitney u test). Treatment neurons in the striatum (Table 1). of rats with MK801, 0.3 mg͞kg per d (n ϭ 10), memantine, 24 Morphological analysis of the neurodegenerative process in- mg͞kg per d (n ϭ 4), or CPP, 24 mg͞kg per d (n ϭ 5), over 28 duced by 3NP in rat striatum by means of light and electron days, did not cause neurological impairment or mortality. Treat- microscopy performed 1 and 2 weeks after initiation and 3 days ment with 3NP, 12 mg͞kg per d, and MK801, memantine, or CPP after termination of treatment with 3NP revealed that initial reduced striatal volume by 12%, 11.5%, and 13%, respectively changes consisted of darkening of neuronal cytoplasm and the and enhanced loss of neuronal density in the striatum by 24%, appearance of cytoplasmic vacuoles that seemed to derive from 26%, and 27.5%, respectively. A total of 497,229 striatal neurons the endoplasmic reticulum (Fig. 1B). The chromatin and the degenerated after systemic administration of 3NP, 12 mg͞kg per nuclear membrane were preserved (Fig. 1B). At more advanced d and vehicle, as opposed to 1,413,014 neurons lost after stages, nuclear chromatin formed clumps that attached to the treatment with 3NP and MK801, 1,423,599 neurons lost af- nuclear membrane, the cytoplasm became darker, and the cells ter treatment with 3NP and memantine, and 1,514,060 lost after shrank (Fig. 1C). No ultrastructural changes were detected in the treatment with 3NP and CPP. Treatment with MK801, 0.3 mg͞kg striatum of vehicle-treated rats (Fig. 1A). per d, memantine, 24 mg͞kg per d, or CPP, 24 mg͞kg per d, alone To explore whether NMDA antagonists affect slowly pro- did not affect survival of neurons in the striatum (Table 1). gressing striatal neurodegeneration, noncompetitive NMDA To investigate involvement of non-NMDA receptors in patho- antagonists, MK801, 0.3 mg͞kg per d, and memantine, 24 mg͞kg genesis of slowly progressing striatal neurodegeneration, com- per d, or a competitive NMDA antagonist, CPP, 24 mg͞kg per petitive AMPA͞kainate antagonists NBQX, 24 mg͞kg per d, and d, were administered s.c. by means of minipumps for 28 days to MPQX, 24 mg͞kg per d, were coadministered s.c. by means of

Table 2. Dose-response relationship of neurotoxic action of 3NP in the rat striatum after intrastriatal administration and protection against 3NP toxicity by the NMDA antagonist CPP Density of neurons within lesion, Neuronal loss, 3 6 3 Treatment, nmol Striatal lesion volume, mm % NV; mean ϫ 10 ͞mm Ϯ SEM % mean Ϯ SEM % n

Vehicle 0.15 Ϯ 0.02 0 0.135 Ϯ 0.003 100 313 Ϯ 41 0 6 3NP 100 1.95 Ϯ 0.32 25 0.028 Ϯ 0.008 21 217,553 Ϯ 17,004 21 6 3NP 250 5.48 Ϯ 0.52 71 0.023 Ϯ 0.002 17 637,720 Ϯ 56,849 62 6 3NP 500 7.73 Ϯ 0.59 100 0.005 Ϯ 0.001 4 1,024,391 Ϯ 97,970 100 6 CPP 250 (4 h) 0.13 Ϯ 0.03 0 0.135 Ϯ 0.004 100 227 Ϯ 39 0 6 3NP 250 (4 h) 5.60 Ϯ 0.38 100 0.023 Ϯ 0.0025 17 642,024 Ϯ 47,203 100 11 3NP ϩ CPP 250 (4 h) 4.01 Ϯ 0.62* 72 0.047 Ϯ 0.0045*** 35 346,841 Ϯ 47,148*** 54 11 CPP 250 (7 d) 0.14 Ϯ 0.02 0 0.134 Ϯ 0.003 100 237 Ϯ 42 0 6 3NP 250 (7 d) 5.84 Ϯ 0.69 100 0.019 Ϯ 0.0025 14 682,330 Ϯ 86,547 100 5 3NP ϩ CPP 250 (7 d) 3.46 Ϯ 0.72* 59 0.037 Ϯ 0.003** 28 338,137 Ϯ 72,747* 50 5

Morphometric analyses revealed that the striatal volume in rats subjected to microinjections of vehicle, 3NP, or CPP did not signiﬁcantly differ from each other. Ͻ Ͻ Ͻ Densities of neurons in nonlesioned parts of the striatum also did not signiﬁcantly differ from each other. *, P 0.05, **, P 0.01, ***, P 0.001 vs. rats treated PHARMACOLOGY with 3NP, 250 nmol (Student’s t test).

Ikonomidou et al. PNAS ͉ November 7, 2000 ͉ vol. 97 ͉ no. 23 ͉ 12887 Downloaded by guest on September 28, 2021 Table 3. Effect of the NMDA antagonist CPP and the AMPA͞kainate antagonist NBQX on neurodegeneration in the hippocampal CA3 subﬁeld after traumatic brain injury Density of pyramidal cells in the Density of pyramidal cells in the

Treatment, hippocampal CA3 subﬁeld, Nv, hippocampal CA3 subﬁeld, Nv, time (h) ipsilateral; mean ϫ 106͞mm3 Ϯ SEM contralateral; mean ϫ 106͞mm3 Ϯ SEM Neurons, mean Ϯ SEM % n

Sham 0.1899 Ϯ 0.0019 0.1891 Ϯ 0.0020 123,435 Ϯ 1,235 100 14 Vehicle 0.1343 Ϯ 0.0024 0.1899 Ϯ 0.0018 85,852 Ϯ 1,534 70 14 CPP, Ϫ2, Ϫ1, 0 0.1579 Ϯ 0.0059 0.1887 Ϯ 0.0016 102,635 Ϯ 3,835ϩϩϩ 83 8 CPP, 1, 2, 3 0.1088 Ϯ 0.0144 0.1883 Ϯ 0.0023 66,368 Ϯ 8,784*** 54 8 CPP, 4, 5, 6 0.1162 Ϯ 0.0051 0.1879 Ϯ 0.0018 69,720 Ϯ 3,061*** 56 9 CPP, 7, 8, 9 0.1233 Ϯ 0.0033 0.1839 Ϯ 0.0016 73,981 Ϯ 1,980** 60 10 CPP, 10, 11, 12 0.1313 Ϯ 0.0068 0.1876 Ϯ 0.0013 84,032 Ϯ 4,352 68 6 NBQX, Ϫ2, Ϫ1, 0 0.1871 Ϯ 0.0043 0.1875 Ϯ 0.0017 115,992 Ϯ 2,666ϩϩϩ 94 8 NBQX, 1, 2, 3 0.1694 Ϯ 0.0035 0.1883 Ϯ 0.0019 106,720 Ϯ 2,205ϩϩϩ 86 8 NBQX, 4, 5, 6 0.1624 Ϯ 0.0018 0.1891 Ϯ 0.0025 100,676 Ϯ 1,116ϩϩϩ 82 8 NBQX, 7, 8, 9 0.1561 Ϯ 0.0016 0.1877 Ϯ 0.0019 96,774 Ϯ 1,001ϩϩϩ 78 7 NBQX, 10, 11, 12 0.1412 Ϯ 0.0021 0.1882 Ϯ 0.0018 86,153 Ϯ 1,281 70 8 Sham ϩ CPP 0.1885 Ϯ 0.0027 0.1892 Ϯ 0.0013 120,650 Ϯ 1,742 98 8 Sham ϩ NBQX 0.1888 Ϯ 0.0034 0.1885 Ϯ 0.0023 120,045 Ϯ 2,169 97 8

Treatment with CPP, 3 ϫ 30 mg͞kg, and NBQX, 3 ϫ 30 mg͞kg, was initiated 2 h before or 1, 4, 7, or 10 h after traumatic cortex injury. To quantitatively assess neuronal loss in the hippocampal CA3 subfield in Fischer 344 rats 3 days after injury, a stereological disector technique was used to estimate Nv. Morphometric analysis revealed that the volume of the hippocampal CA3 subfield in rats subjected to traumatic brain injury and treatment with CPP or NBQX did not significantly differ from that in rats subjected to brain trauma and vehicle and ranged from 0.61 to 0.64 mm3. The differences between vehicle- and drug-treated rats in the numbers of neurons remaining in the hippocampus after injury were analysed statistically by means of Student’s t test. **,ϩϩ, P Ͻ 0.01; ***,ϩϩϩ, P Ͻ 0.001 vs. vehicle-treated rats. Stars indicate significant increase in neurodegeneration, while crosses indicate neuroprotection. Monitoring of blood pressure and arterial blood gases in rats subjected to head trauma and systemic treatment with either CPP (n ϭ 8) or NBQX (n ϭ 12) showed no evidence of cardiorespiratory compromise.

minipumps for 28 days to rats subjected to treatment with 3NP, Degeneration in the striatum induced by 3NP, 12 mg͞kg per d, 12 mg͞kgperdor24mg͞kg per d. NBQX and MPQX prevented also was reduced by NBQX and MPQX (Table 1). neuronal degeneration in the striatum induced by 3NP, 24 mg͞kg per d, (Table 1) and led to an improvement of neurologic Intrastriatal 3NP and Glutamate Antagonists. To determine whether outcome (score 0.71 Ϯ 0.29, n ϭ 7 for 3NP ϩ NBQX and 1.37 Ϯ NMDA antagonists affect rapidly progressing striatal neurodegen- 0.32, n ϭ 8 for 3NP ϩ MPQX vs. 2.41 Ϯ 0.21 for 3NP ϩ vehicle, eration, CPP, 250 nmol, was coadministered with 3NP, 250 nmol, n ϭ 19; P Ͻ 0.005 and P Ͻ 0.01, Mann–Whitney u test). into the striatum, and the extent of the damage was assessed

Fig. 2. Light microphotographs demonstrating the effect of the NMDA antagonist CPP on slowly progressing neurodegeneration induced in the striatum by systemic administration of 3NP and in the hippocampus by traumatic brain injury. (A) Morphology of striatum after treatment with 3NP, 12 mg͞kg per d over 28 days, 3 days after termination of treatment. No obvious neurodegeneration can be detected in the striatum; the spiny neurons have normal appearance. (B) Profound neuronal loss and predominance of glia in the striatum 3 days after termination of treatment with 3NP, 12 mg͞kg per d, and CPP, 24 mg͞kg per d over 28 days. Large-size striatal neurons are relatively preserved. (C) Hippocampal pathology in the CA3 subfield 3 days after traumatic brain injury. Dark argyrophylic profiles indicate ongoing degeneration in pyramidal layer. Intact pyramidal cells with prominent nuclei are also present. (D) The effect of treatment with CPP, 3 ϫ 30 mg͞kg given i.p. 1, 2, and 3 h after trauma is shown. Widespread degeneration of pyramidal neurons predominates. Magnifications: A and B, ϫ60 (cresyl violet stain); C and D, ϫ80 (Fink and Heimer stain).

12888 ͉ www.pnas.org Ikonomidou et al. Downloaded by guest on September 28, 2021 stereologically. 3NP caused widespread neurodegeneration in the striatum characterized ultrastructurally by swelling of dendrites, neuronal somata, and cytoplasmic organelles, early breakdown of cytoplasmic and nuclear membranes, and elimination of cell rem- nants by inflammatory cells. CPP prevented morphological se- quelae and reduced the lesion volume induced by 3NP by 28% (Table 2). A total of 642,024 striatal neurons degenerated after intrastriatal injection of 3NP, 250 nmol, as opposed to 346,841 neurons degenerating after treatment with 3NP and CPP (Table 2).

Traumatic Brain Injury and Glutamate Antagonists. In rats subjected to traumatic brain injury, rapidly developing neurodegeneration occurs in the cortex adjacent to the site of injury, whereas slowly progressing degeneration occurs in the hippocampal CA3 subfield distant to the site of injury. To evaluate whether NMDA antagonists differentially affect neurodegeneration triggered by traumatic brain injury, rats were subjected to cortex trauma, and the NMDA antagonist CPP, 30 mg͞kg, was administered at either 2, 1, or 0 h before trauma, 1–3 h, 4–6 h, 7–9, or 10–12 h after trauma, and the extent of cortical as well as hippocampal degeneration was evaluated 3 days after trauma. When treatment with CPP was initiated at 2 h before trauma, the volume of the damage in the parietal cortex was reduced by 37% (7.17 Ϯ 0.78 mm3, n ϭ 8 vs. 11.26 Ϯ 1.18 mm3, n ϭ 14 in vehicle-treated rats; P Ͻ 0.05, Student’s t test). When treatment was delayed by1hormore, no significant difference in the volume of cortical damage was noted between CPP- and vehicle-treated rats (the volume of the cortex damage varied between 10.62 and 11.78 mm3). Damage in the CA3 subfield was decreased by treatment with CPP commencing 2 h before trauma, but was enhanced by treatment commencing 1, 4, and7haftertraumatic injury to the cortex (Table 3). A total of 37,583 pyramidal neurons degenerated after traumatic brain injury in the CA3 subfield of vehicle-treated rats, as opposed to 57,067 neurons degenerating after treatment with CPP commencing 1 h, 53,715 when treatment started at 4 h, and 49,454 when treatment was initiated7haftertrauma. Treat- ment with CPP, initiated as late as 10 h after trauma, did not affect the damage in the hippocampal CA3 subfield (Figs. 2 and 3). CPP, ϫ ͞ Fig. 3. Effect of CPP on morphology of the hippocampal CA3 subfield in rats 3 30 mg kg i.p., did not induce neuronal death in the hippocam- subjected to traumatic cortex injury. CPP was administered i.p. at a dose of 30 pus of rats subjected to sham surgery or in the hippocampus on the mg͞kg at 2, 1, and 0 h before, 1, 2, and 3 h, 4, 5, and 6 h, 7, 8, and 9 h, or 10, 11, ͞ site contralateral to traumatic injury (Table 3). The AMPA kainate and 12 h after traumatic injury. Nvs of pyramidal cells in the hippocampal CA3 antagonist NBQX reduced the volume of the damage in the parietal subfield were estimated between rostro-caudal coordinates 9.61 and 12.21 ac- cortex on antecedent treatment by 39% (6.84 Ϯ 0.89 mm3, n ϭ 8; cording to the stereotaxic atlas of Swanson (10) using the stereologic disector P Ͻ 0.05, Student’s t test), but had no effect on the volume of method (12, 13). Traumatic brain injury in vehicle-treated rats resulted in signif- cortical damage on delayed treatment. NBQX also prevented icant reduction of neuronal densities in the ipsilateral (right, R) hippocampal CA3 subfield and had no effect on densities of pyramidal cells on contralateral side hippocampal neurodegeneration when administered 2 h before, as (left, L). Two-way ANOVA revealed that treatment with CPP commencing 2 h wellas1,4,or7haftertraumatic injury (Table 3). NBQX had no before traumatic injury prevented reduction of numerical density of pyramidal protective effect when treatment was initiated at 10 h (Table 3). cells in the ipsilateral CA3 subfield [F-2h(1,12) ϭ 6.97, P Ͻ 0.001, n ϭ 8] as compared Only 16,715 pyramidal neurons degenerated after traumatic brain with vehicle-treated rats (n ϭ 14). Treatment with CPP starting at1h[Fϩ1h(1,12) ϭ injury in the CA3 subfield of NBQX-treated rats when treatment 5.56, P Ͻ 0.001, n ϭ 8],4h[F4h(1,12) ϭ 3.43, P Ͻ 0.001, n ϭ 9],or7h[F7h(1,12) ϭ was initiated 1 h, 22,759 when treatment was started 4 h, and 26,661 1.31, P Ͻ 0.005, n ϭ 10] after traumatic injury significantly enhanced decrease of when treatment was initiated7haftertrauma. NBQX did not numerical density of pyramidal cells in the ipsilateral CA3 subfield, whereas ϭ ϭ induce neuronal death in the hippocampus of rats subjected to sham treatment commencing after 10 h was ineffective [F10h(1,12) 0.06, NS, n 6]. surgery or in the hippocampus on the site contralateral to traumatic injury (Table 3). Monitoring of blood pressure (systolic and dia- stolic), arterial blood gases (pH, P O ,PCO , bicarbonate), and antagonist CPP also exacerbated slowly progressing neurodegen- a 2 a 2 eration in the hippocampus triggered by traumatic cortex injury body temperature in rats subjected to traumatic brain injury showed when administered between 1 and7hafterinjury. In contrast, no evidence of cardiorespiratory compromise or hypothermia. neurons that degenerated rapidly after exposure to traumatic injury Discussion to the cortex, responded favorably to antecedent treatment with CPP. Thus, slowly progressing neurodegeneration triggered by two NMDA antagonists MK801, memantine, and CPP exacerbated different mechanisms was enhanced by NMDA antagonists. slowly progressing neurodegeneration in the striatum produced by The AMPA antagonists NBQX and MPQX prevented both 3NP. In contrast, neurons that degenerated rapidly after microin- rapid and slowly progressing neuronal death induced by 3NP or jection of 3NP into the striatum responded favorably to treatment traumatic brain injury. These observations suggest that gluta- with the NMDA antagonist CPP. The exacerbation is unlikely to mate differentially determines the fate of neurons endangered to depend on compromise of oxidative phosphorylation by NMDA die slowly after brain injuries. It may save neurons by activating

antagonists, because NMDA antagonists did not block activity of NMDA receptors or destine neurons to die by activating PHARMACOLOGY succinate dehydrogenase in neuronal cultures (14). The NMDA AMPA͞kainate receptors.

Ikonomidou et al. PNAS ͉ November 7, 2000 ͉ vol. 97 ͉ no. 23 ͉ 12889 Downloaded by guest on September 28, 2021 The opposite response to NMDA antagonists of rapid and slowly Alternatively, NMDA and AMPA receptor antagonists may progressing neurodegeneration raises questions as to the mecha- differently influence activity of neuronal populations by decreas- nisms involved. High levels of energy compromise or profound ing the input to or output from GABAergic interneurons and elevation of extracellular glutamate concentrations after traumatic enhancing input to or output from cholinergic interneurons to injury lead to depolarization of neuronal membranes, relief of the promote or prevent neuronal death (28, 29). voltage-dependent Mg2ϩ block of NMDA receptor channels, and The apparently paradoxical promotion of neurodestruction by NMDA receptor-mediated excitotoxicity (15). Under such condi- NMDA antagonists on one hand and neuroprotective action of tions NMDA antagonists are neuroprotective. In contrast, slowly AMPA͞kainate antagonists on the other in slowly progressing progressing neurodegeneration induced by low level of energy neurodegeneration in the striatum subjected to systemic 3NP compromise or traumatic injury at sites distant to the impact has and in the hippocampus subjected to traumatic injury suggest been suggested to evolve in a programmed fashion that involves that such mechanisms may be operative in vivo. activation of caspases (16). Low-intensity stimulation of NMDA Our findings have implications for clinical trials with NMDA receptors increases intracellular Ca2ϩ concentration and protects antagonists, which were initiated in humans based on neuropro- cells from caspase-mediated death (17). In murine cortical neurons, tection that has been demonstrated in rodent models of stroke and elevation of intracellular Ca2ϩ protects against ischemic apoptosis.§ trauma on antecedent treatment. In patients undergoing cardio- Thus, decrease of intracellular Ca2ϩ concentration caused by pulmonary bypass and subjected to antecedent treatment with the blockade of Ca2ϩ-permeable NMDA channels may be one mech- NMDA antagonist remacemide starting at 4 days before surgery, a anism contributing to enhancement of slowly progressing neuronal beneficial effect on neuropsychomotor performance has been death by NMDA antagonists. documented (30). These observations are in agreement with our Stimulation of NMDA but not AMPA͞kainate receptors has findings showing that in rats subjected to antecedent treatment with been shown to enhance mRNA levels of neurotrophins in the CPP neuronal loss in the hippocampus after traumatic brain injury injured brain (18, 19). Synthesis of neurotrophins increases in the can be mitigated. It is also in agreement with the observation that brain after exposure to neurotoxins (20) and in the hippocampus MK801 and memantine prevent learning deficits in rats caused by after traumatic brain injury (21). Thus, it appears feasible that acute damage of the hippocampus triggered by the NMDA agonist prevention of neurotrophin synthesis by NMDA antagonists may be quinolinate (31). In no other clinical trial, including those in stroke deleterious for neuronal survival following injuries. Neurotrophins and head or spinal cord trauma, in which delayed treatment such as brain-derived neurotrophic factor (BDNF), NT3, or NT4͞5 regimens with NMDA antagonists were chosen, could neuropro- possess glutamate-like depolarizing properties in mature neurons tective effects be confirmed so far (1) and in fact, in some trials, (22). Neurotrophins enhance NMDA-mediated excitotoxic degen- increased mortality prompted termination (32, 33). In chronic eration in murine neurons (23) and may protect either against neurodegenerative disorders, such as Huntington’s or Parkinson’s degeneration induced by serum deprivation in vitro (23), against disease, symptomatological improvement with NMDA antagonists hippocampal damage induced by ischemia or spinal cord damage has been documented, but increases of survival or slowing of induced by traumatic injury (24, 25), or against damage of substan- disease progression have not been reported so far (2). Symptom- tia nigra produced by mitochondrial toxins (26). It can be specu- atological improvement seen in chronic neurodegenerative disor- lated that glutamate acting via NMDA receptors may serve a ders with NMDA antagonists can be explained by their interaction trophic function resembling that of neurotrophins in mature brain. with glutamate receptors in the basal ganglia that are involved in Antagonists of neurotransmitter receptors trigger in vivo transmitting the symptoms of the disease to respective motor compensatory elevation of synthesis and͞or release of agonists centers and not necessarily by prevention of degeneration of striatal from presynaptic endings (27). It can be hypothesized that when or nigral neurons (34, 35). NMDA receptors are selectively blocked by antagonists such as In light of our current findings, caution should be exercised MK801, memantine, or CPP, glutamate will activate AMPA͞ when using NMDA antagonists as monotherapy in humans kainate receptors, for as long as imbalance in the NMDA system suffering from brain injuries or progressive neurodegenerative continues, and will promote neuronal death. Selective blockade disorders with unknown mechanism of neuronal death. If our of AMPA͞kainate receptors by antagonists such as NBQX or observations are valid for humans, our conclusions may explain MPQX will divert action of glutamate toward NMDA- why clinical trials have failed to identify neuroprotective effects dependent mechanisms and promote survival (13). of NMDA antagonists.

This work was supported by Grant 01K095151 from the Bundesminis- §Babcock, D. J., Gottron, F. J. & Choi, D. W. (1999) Soc. Neurosci. Abstr. 25, 2103. terium fu¨r Bildung und Forschung (BMBF).

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