Neuroprotective Effects of Progesterone on Damage Elicited by Acute Global Cerebral Ischemia in Neurons of the Caudate Nucleus

Archives of Medical Research 33 (2002) 6–14

ORIGINAL ARTICLE

Neuroprotective Effects of Progesterone on Damage Elicited by Acute Global Cerebral Ischemia in Neurons of the Caudate Nucleus Miguel Cervantes,a María Dolores González-Vidal,b Rodrigo Ruelas,b Alfonso Escobarc and Gabriela Moralíb a

Laboratorio de Neurofarmacología, Centro de Investigación Biomédica de Michoacán, Instituto Mexicano del Seguro Social (IMSS), Morelia, Michoacán, Mexico b Unidad de Investigación Médica en Farmacología, Centro Médico Nacional Siglo XXI (CMN-SXXI), IMSS, Mexico City, Mexico c Departamento de Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico Received for publication January 9, 2001; accepted May 25, 2001 (01/007).

Background. In addition to the hippocampus, the dorsolateral caudate nucleus (CN) and the pars reticularis of the substantia nigra (SNr) are among the most vulnerable brain areas to ischemia. A possible association of the neuronal injury in these two subcortical nuclei has been proposed, the primary damage affecting the CN GABAergic neurons innervating the SNr, and secondarily the SNr neurons as a result of an imbalance of GABAergic and glutamatergic input to the SNr. Progesterone (P4) exerts a GABAergic action on the central nervous system (CNS) and is known to protect neurons in the cat hippocampus from the damaging effect of acute global cerebral ischemia (AGCI). The effects of AGCI on the neuronal populations of the CN and SNr, in addition to the possible neuroprotective effects of P4, were assessed in cats in the present study. Methods. Ovariectomized adult cats were treated subcutaneously (s.c.) with either P4 (10 mg/kg/day) or corn oil during the 7 days before and 7 days after being subjected to a period of AGCI by 15 min of cardiorespiratory arrest followed by 4 min of reanimation. After 14 days of survival, animals were sacrificed and their brains perfused in situ with phosphate-buffered 10% formaldehyde for histologic examination. Results. ACGI resulted in an intense glial reaction in the CN and a significant loss (43%) of medium-sized neurons of the CN, but no difference was found in the densities of SNr neurons between controls and ischemic oil- and P4-treated cats. Progesterone treatment completely prevented CN neuronal loss. Conclusions. The overall results point to the higher vulnerability of CN neurons to ischemia as compared to neurons in the SNr and show the protective effects of P4 upon CN neuronal damage after ischemia. © 2002 IMSS. Published by Elsevier Science Inc. Key Words: Global cerebral ischemia, Neuroprotection, Caudate nucleus, Progesterone, Cat.

Introduction Acute global cerebral ischemia (AGCI) triggers a series of pathophysiologic phenomena that result in acute, maturational, and delayed neuronal death in specific, highly vulnerable brain structures (1–16) that include the following:

Address reprint requests to: Gabriela Moralí, Ph.D., Unidad de Investigación Médica en Farmacología, CMN-SXXI, IMSS, Apdo. Postal 73-032, 03020, México, D.F., México. Tel.: (52) (55) 5687-8606; FAX: (52) (55) 5761-0952; E-mail: [email protected]

the pyramidal neurons of hippocampal CA subfields; pyramidal neurons in cerebral cortex layers 3 and 5; Purkinje cells in the cerebellum, and middle- and small-sized neurons in the dorsolateral striatum (6,17–22). Several mechanisms have been associated with the high vulnerability of these neuronal types, including abundance of glutamatergic or dopaminergic innervation (4,6,8,23,24) and the content of certain metallic compounds (25) among others. In particular, the excessive glutamatergic and dopaminergic activity have been shown to be involved in ischemia-induced neuronal damage in the striatum (26–28), where the cytotoxic

0188-4409/02 $–see front matter. Copyright © 2002 IMSS. Published by Elsevier Science Inc. PII S0188-4409(01)00 3 4 7 - 2

Progesterone Neuroprotection in Caudate Nucleus

effect of excessive dopamine (DA) seems to be mediated by D2 receptors and free oxygen radicals resulting from the metabolism of the excessive amounts of DA released during ischemia and biotransformed during reperfusion (29–31). Selective damage to the CA1 pyramidal neurons after ischemia appears to result from an imbalance between excitatory and inhibitory influences (32,33). Thus, agents reducing excitatory aminoacid neurotransmission (34–37) or increasing GABAergic inhibitory neurotransmission (38–46) protect CA1 pyramidal neurons from the ischemia damaging effect. Neuroprotective compounds that are effective in brain structures such as the hippocampus, in which dopaminergic activity is not a main component of pathophysiologic mechanisms of neuronal damage, may exhibit different neuroprotective effects in the striatum where dopaminergic mechanisms of neuronal damage are important. Nevertheless, the GABAergic inhibitory influence directly exerted by CN interneurons on medium-sized caudate neurons (47–49) in addition to the GABAergic influence on nigrostriatal dopaminergic neurons (50,51) may support a possible GABAergicmediated neuroprotective effect such as that suggested for progesterone (P4) (52–55). An association has been proposed to exist between the neuronal damage in the dorsolateral part of the caudate nucleus (CN) and the pars reticularis of the substantia nigra (SNr), where the primary damage affects the CN GABAergic neurons innervating the SNr and secondarily the SNr neurons as a result of an imbalance of GABAergic and glutamatergic input to the SNr (56–59). Thus, ischemic neuronal damage in the SNr may be prevented or reduced by increasing the GABAergic activity (40,41,45). In previously reported data (53), the neuroprotective effects of P4 on neuronal damage in the cat hippocampus were explained by an enhancement of the GABAergic inhibitory influence exerted by this steroid either per se or through its biotransformation in the brain. Steroids derived from the biotransformation of progesterone interact with specific recognition sites in the GABAA receptor increasing the GABAergic neurotransmission (60–65). Furthermore, neurologic assessment of consciousness, sensory, motor, autonomic, and behavioral conditions in ischemic, progesterone-treated cats showed significantly lower damage as compared to that in ischemic, vehicle-treated cats (53). These findings suggest that the neuroprotective effect of progesterone is also exerted in other brain structures in addition to the hippocampus. Thus, an analysis of the neuronal populations of the substantia nigra and the dorsolateral striatum was considered to be of interest, in view of their functional and neuroanatomic relations. Materials and Methods The experimental protocol was approved by the Scientific Research Committee (March 29, 1996). Brain tissue samples used for histologic analysis of the caudate nucleus and

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substantia nigra were obtained from cats included in a previous study (53). Assignment of cats to the three experimental groups as well as the experimental procedures to which they were subjected was previously described (53). In brief, subjects included 18 adult ovariectomized female cats (2.5– 3.2 kg body weight [b.w.]) randomly allotted to one of three groups and given daily subcutaneous (s.c.) injections of either vehicle (corn oil, 0.5 mL/kg/day, groups 1 and 2) or progesterone (10 mg/kg/day in corn oil, group 3) during 7 days. This dose of progesterone allows the achievement and maintenance of stable blood levels of this steroid above physiologic concentrations (53). Blood samples were obtained under light anesthesia from a hind leg in all cats at days 0, 2, 4, and 7 of treatment to measure circulating P4 by an automated immunoassay. The serum was separated immediately and stored at 4C until assayed. Progesterone was measured by a chemiluminescent enzyme immunoassay using commercial kits (IMMULITE Progesterone, Diagnostic Products Corporation, Los Angeles, CA, USA). The detection limit for P4 was 0.09 ng/ mL and the intra- and interassay coefficient of variation was 6 and 8.5%, respectively. On day 7, each cat of groups 2 and 3 was submitted to a 15-min period of acute global cerebral ischemia as a result of cardiorespiratory arrest (CRA), followed by reanimation within 4 min, according to a model of AGCI previously described (53,66–68). Animals in group 1 were subjected to sham procedures only. Experiments were carried out under controlled conditions including halothane anesthesia, assisted mechanical ventilation, blood pressure, blood pH, base excess, PaO2, PaCO2, glucose, and body temperature (53,68) as follows: cats were anesthetized for prearrest surgery with 4% halothane in oxygen; pancuronium bromide, 0.3 mg/kg, was administered intravenously (i.v.) through a sterile venous catheter placed in a hind leg, and endotracheal intubation was performed and assisted ventilation with 1.5% halothane in oxygen (Bird MarkVIII ventilator) was begun to maintain anesthesia and PaCO2 between 30 and 35 mmHg. After previous skin infiltration with 1 mL of 2% lidocaine, a small incision (1 cm) was made in the neck and in the groin. A sterile catheter was inserted through the jugular vein to guide the tip of a wire (0.75 mm in diameter) into the right atrium; its precise location was confirmed by cavitary electrocardiogram (EKG). Another sterile catheter was inserted into the right femoral artery for continuous monitoring of mean arterial pressure (MAP). On completion of surgery, neck and groin wounds were sutured and covered. Halothane administration was interrupted and 1 min later ventricular fibrillation was induced in cats of groups 2 and 3 by passing alternating current (60 Hz, 20 V, 5–10 sec) from the tip of the atrial wire to a subcutaneous electrode placed at the apex until MAP showed a sudden decrease to 10 mmHg. Then, mechanical ventilation was stopped and the tracheal cannula was occluded. Five min. after the beginning of the cardiac arrest, the atrial wire was removed.

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Cervantes et al./ Archives of Medical Research 33 (2002) 6–14

Cardiac arrest and interruption of mechanical ventilation were maintained for 15 min. Cardiopulmonary resuscitation was initiated at the end of this period as follows: mechanical ventilation with FiO2  1; external cardiac massage to increase and maintain MAP at 90 mmHg, and administration of sodium bicarbonate, 1 mEq/kg i.v., and epinephrine hydrochloride, 15 g/kg i.v. Defibrillation (Mennen Cardiopak model 936, Mennen Greatbatch Electronics, Inc., Clearance, NY, USA) by means of a 20 J DC shock applied between two chest paddles placed on the shaved lateral chest walls was first attempted 2 min after initiating cardiopulmonary resuscitation. When unsuccessful, additional sodium bicarbonate and epinephrine hydrochloride were administered and defibrillation was repeated until successful when MAP 90 mmHg was reached and maintained. Immediately after defibrillation, atropine sulfate, 50 g/kg i.v. and lidocaine hydrochloride, 1 mg/kg i.v. were administered when needed to assist stabilization of cardiac sinusal rhythm. Cardiopulmonary resuscitation within a period no longer than 4 min was a necessary condition for animals to be included in the study. EKG (lead II) was continuously monitored through subcutaneous needle electrodes. Esophageal temperature was kept at 37.0–37.5C. A sample (1 mL) of arterial blood was drawn 15 min prior to CRA, at 5 and 20 min, and at 1, 2, and 4 h following CRA or sham maneuvers to determine pH, PaO2, PaCO2, bicarbonate, base excess (pH-Blood GasCIBA Corning 2381 model, Ciba Corning de México, S.A. de C.V., México, D.F.), and glucose along the experiment. Changes in their pre-arrest values were promptly corrected through the administration of sodium bicarbonate or ventilation adjustment. Values of PaCO2 30–35 mmHg and pH 7.30–7.35 were maintained until reversal of neuromuscular blockade. Assisted ventilation was maintained at FiO2  1 for 1 h after CRA or sham procedures and FiO2  0.4 afterward. Neuromuscular blockade (pancuronium bromide, 0.5 mg/kg/h i.v.) was maintained until 6 h after CRA. Cats in group 1 received no alternating current via the wire inserted through the jugular venous catheter to the atrium and mechanical ventilation was not stopped; thus, these cats were subjected neither to cardiorespiratory arrest nor resuscitation maneuvers but only to sham procedures including neuromuscular blockade and assisted ventilation for 6 h. At this time, the cats were allowed to recover from neuromuscular blockade until normal spontaneous respiratory activity was resumed. Neostigmine methylsulfate 0.06 mg/ kg was administered i.v. to reverse neuromuscular blockade and atropine sulfate, 0.04 mg/kg i.v. was administered to prevent bradycardia. Arterial and atrial cannulas were removed under halothane anesthesia and the animals were extubated. After extubation, each cat was placed in a cage at a temperature of 25C until 24 h after resuscitation. On the following days, cats were allowed to drink milk and water and eat tuna fish paste. If needed, 50 mL/kg/day maintenance fluid was injected s.c.

Progesterone or vehicle treatment was continued for an additional 7 days following CRA or sham procedures. Each cat was subjected to daily neurologic evaluations in a blind manner, assessing a number of neurologic parameters such as level of consciousness, respiration, cranial nerves, and spinal reflexes as well as postural, locomotor, and behavioral reactions according to the procedure designed by Todd et al. (66). Points were assigned to each neurologic alteration and added together to obtain a neurologic deficit score ranging from 0 to 100 (score 0, normal neurologic condition; score 100, maximal neurologic deficit). On survival day 14, the cats were deeply anesthetized with pentobarbital (35 mg/kg i.p.), the chest was opened, the right auricle incised, and a 14-gauge needle inserted into the left ventricle for perfusion. Transcardiac perfusion began with 400 mL saline, followed by 800 mL 10% phosphate-buffered formaldehyde and 300 mL Clarke fixative (ethanol-acetic acid 3:1 v/v) (69). Following perfusion, the brains were removed and immersed in the same fixative for at least 7 days prior to histologic processing. Brains were then cut into 3-mm coronal slices, dehydrated, and embedded in paraffin. Semiserial 10-m sections were sampled from the caudate nucleus and the substantia nigra, and stained with Klüver-Barrera technique with Luxol fast blue and cresyl violet (70). For each brain, five sections through the central portion of the caudate nucleus, located between A15.0 and A17.0, i.e., 15–17 mm anterior to the interaural line according to the atlas of Snider and Niemer (71), and five sections of the substantia nigra at the level of the emergence of the oculomotor nerve (cranial nerve III) at the ventral mesencephalon between A4.5 through A5.5, i.e., 4.5–5.5 mm anterior to the interaural line, were analyzed for cell counting. The number of surviving medium-sized (17–25 m in diameter) neurons in at least six microscopy fields measuring 450 m in diameter, located in the dorsolateral part of the caudate nucleus in each section, was counted and averaged. Only neurons showing normal morphology and visible nucleolus were counted. The number of surviving large (15–42 m the longest diameter) and small (10–15 m in diameter) neurons of the reticulata and globular types (72), in at least six microscopy fields of 450 m in diameter located in the pars reticularis of the substantia nigra in each section, was counted and averaged. Only neurons showing normal morphology, abundant Nissl material, and visible nucleolus were counted. Neurons that had shrunken cell bodies with surrounding empty spaces were excluded. Sections were examined in a blind fashion under light microscopy at a magnification of 400. The average numbers of neurons in the various microscopy fields were evaluated in each animal. Analysis of variance and Duncan tests were used to compare body weight, progesterone levels, MAP, blood glucose, pH, blood gases, and base excess values under the different experimental conditions. Mann-Whitney U test was used to compare neurologic deficit scores between proges-

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Table 1. Values (mean SD) of the physiologic variables recorded in cats under different experimental conditions Group Body weight (kg) Serum progesterone (ng/mL) Day 0 Day 2 Day 4 Day 7 pH Basal 20 min 14 h Base excess (mEq/lt) Basal 20 min 14 h PaCO2 Basal 20 min 14 h PaO2 Basal 1h 24 h Glucose (mg/dL) Basal 1030 min 14 h MAP Basal 30 min 14 h

SHAM

ISQ  VEH

ISQ  P4

2.9 0.4

2.9 0.3

3.0 0.6

2.1 0.7 1.5 0.3 2.5 0.7 1.9 0.5

2.3 0.6 1.5 0.7 1.8 0.6 2.8 0.7

1.9 0.8 122.0 27.3b 146.3 35.2b 193.5 70.2b

7.36 0.02 7.36 0.15 7.37 0.20

7.38 0.08 7.15 0.30b 7.36 0.20

7.36 0.08 7.17 0.10b 7.35 0.13

3.0 0.1 11.0 1.6 9.1 0.6

11.0 2.0 22.0 4.0b 11.2 4.1

12.0 3.0 17.0 3.0b 9.0 2.2

26.0 2.0 15.0 8.0 21.0 8.0

21.0 6.0 37.0 13.0a 23.0 11.0

21.0 8.5 30.0 10.0a 24.0 8.0

84.0 13.0 90.0 15.0 102.5 20.2

120.0 45.0 233.1 90.5a 129.7 39.0

124.0 64.0 219.5 111.0a 139.2 56.4

130 21 150 25 165 21215 50

136 31 208 48a 185 92227 36

127 54 268 67a 189 33204 61

95 5 110 28 120 14140 14

110 22 160 28a 119 18136 20

107 22 134 24a 109 22126 22

p 0.05; bp 0.01 as compared to the sham group. Duncan test.

a

terone- and vehicle-treated cats. Values of number of neurons per microscopy field in each cat in each experimental group were expressed as median and range. Statistical analysis was done using Kruskal-Wallis test followed by MannWhitney U tests for comparison of number of each type of neurons counted in the caudate nucleus and in the substantia nigra, among the groups (73,74). Results Values of body weight were not significantly different among the three groups of cats, i.e., sham (group 1), vehicle (group 2)-, and P4 (group 3)-treated groups submitted to ischemia (Table 1). Data on the different variables relevant for the experimental model of acute global cerebral ischemia and on neurologic outcome under vehicle or P4 treatment were previously reported in the same experimental subjects included in the present study (53) and are summarized in Tables 1 and 2. Serum levels of P4 in female cats on day zero, prior to initiating vehicle or progesterone treatment, were similarly low in groups 1, 2, and 3; serum levels of P4 in cats receiving vehicle remained low throughout the 7 days of treatment, and daily s.c. administration of progesterone resulted in a gradual increase in serum levels of P4

that reached 193.5 70.2 ng/mL at day 7 after onset of P4 treatment. Blood gases, pH, base excess, and MAP were within physiologic ranges in all cats immediately before CRA or sham procedures and were similar among the sham, the vehicle-treated, and the P4-treated groups. A transient but significant decrease in pH and base excess values (p 0.01) and a significant increase in PaCO2 values (p 0.05) were found 5 and 20 min after the end of resuscitation in both vehicle- and P4-treated cats submitted to ischemia as compared to their pre-CRA values, and to the sham group. However, these values were corrected, so that mean values of the blood components obtained in subsequent determinations from 1–4 h after CRA were not significantly different from those obtained prior to CRA. An increment in PaO2 values (p 0.05) due to assisted ventilation with FiO2  1 during the first hour after CRA was found in both experimental groups (233.1 90.5 and 219.5 111.0 mmHg, respectively). Blood gases, pH, and base excess were within the physiologic range in intact cats submitted to sham procedures. In both groups submitted to ischemia, plasma glucose concentrations significantly increased after resuscitation (Table 1) and remained high throughout the post-CRA pe-

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Cervantes et al./ Archives of Medical Research 33 (2002) 6–14

Table 2. Neurologic deficit scores shown on the first 7 days following ischemia, by cats treated either with vehicle or with progesterone (P4, 10 mg/kg/day) Ischemia  vehicle Md range Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

69.5 49.5 47.0 40.0 31.0 25.0 15.5

5681 2671 1666 1262 857 657 342

Ischemia  P4 Md range 28.0 17.0 16.0 11.5 8.0 5.0 3.0

2342a 1430a 1020a 812a 411a 17 16

p 0.05 as compared to vehicle-treated cats. Mann-Whitney U test.

a

riod; however, differences were not significant between groups when compared at specific times after CRA. In the group submitted to sham procedures, plasma glucose concentrations ranged from 130 21 mg/mL to 215 50 mg/ dL during the entire experimental period, values significantly lower than those of groups 2 and 3 at 10 and 30 min after CRA, but not later. Values of MAP were elevated during the first minute after CRA in the vehicle-treated and P4-treated groups and decreased to values similar to those of the sham group during the remaining experimental period. There were no significant differences in these values when comparisons were made at 1-h intervals after CRA between vehicle- and P4treated groups. Esophageal temperature was maintained at 37.0–37.5C in all cats throughout the experimental phase. Neurologic deficit scores (Table 2) ranged from 56 to 81 points on day 1 post-CRA in the vehicle-treated cats and from 23 to 42 points in the progesterone-treated animals. These scores clearly tended toward a reduction in both vehicle- and P4-treated groups, showing neurologic deficit scores from 12 to 62 and from 8 to 12, respectively, on day 4 and from 3 to 42 and 1 to 6, respectively, on day 7; no further changes were observed on days 8–14 after CRA. Thus, neurologic deficit scores in this period are not shown in Table 2. High neurologic deficit scores in vehicle-treated cats resulted from persistence of abnormal pupil size and light reflex, diminution of facial pain perception, flexor reflex to pain, and orienting reflex to loud clap, as well as from lack of placing paw reflex, on the days following CRA. Neurologic deficit scores were significantly lower (p 0.05) in P4-treated cats on the days following CRA than in vehicletreated cats. Figure 1 shows representative images of the neuronal population in the caudate nucleus of cats subjected to sham procedures (upper image) or subjected to ischemia and treated either with the vehicle (middle image) or with P4 (lower image). In the cats subjected to 17–19 min of ischemia and treated with vehicle, there was a clear reduction in the number of medium-sized neurons in the caudate nucleus as compared to that of cats in the sham group. On the other hand, in progesterone-treated cats subjected to is-

Figure 1. Representative photomicrographs of the neuronal population found in the dorsolateral caudate nucleus of cats subjected to sham procedures (SHAM), and cats subjected to acute global cerebral ischemia under either vehicle (ISCH  VEH) or progesterone (ISCH  P4) treatment. Note the smaller amount of medium-sized neurons in the caudate nucleus of ischemic, vehicle-treated cats as compared to sham cats, and the preservation of neurons in P4-treated cats. Luxol fast blue and cresyl violet. Scale bar, 100 m.

chemia, the neuronal population was equal to that in sham cats. An intense glial reaction was associated with the reduction of the neuronal population in the caudate nucleus of vehicle-treated cats. Numerical data of medium-sized neurons in the caudate nucleus of the different groups of cats are shown in Table 3. In the group treated with vehicle and subjected to ischemia, the number of neurons (Md: 37.6/field; range: 29.5–68.4) was significantly lower (p 0.01) than that of the sham

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Table 3. Number of medium-sized neurons (1725 m diameter) found in microscopy fields of 450 m of diameter in the dorsolateral caudate nucleus of cats under the various experimental conditions Number of neurons/field (Md, range) Sham Ischemia  vehicle Ischemia  P4

66.4 37.6 63.0

51.880.3 29.568.4a 48.673.0b

p 0.01 as compared to the sham group; bp 0.01 as compared to the vehicle-treated group. Mann-Whitney U test.

a

group (Md: 66.4/field; range: 51.8–80.3), amounting to only 57% of the total number of neurons in sham cats taken as 100% (Table 3). In contrast, surviving neurons in the progesterone-treated cats subjected to ischemia (Md: 63.0/field; range: 48.6–73.0) amounted to 95% of the total neuronal population of the sham group (100%) without significant differences between these two groups; numbers of neurons in the caudate nucleus of P4-treated cats were significantly higher (p 0.01) than those of vehicle-treated animals (Table 3). On the other hand, neuronal population in the pars reticularis of the substantia nigra did not differ among groups, as seen in Figure 2 and Table 4.

Discussion In the present study, a carefully controlled experimental model of acute global cerebral ischemia provoked by cardiorespiratory arrest was used. Control of variables (MAP, blood gases, pH, etc.) able to influence the magnitude of ischemic-induced neuronal damage was carried out in a similar manner in each cat from the different experimental groups, as we and other authors have done in similar studies (53,66– 68). In particular, blood glucose increases were similar between P4-treated and vehicle-treated cats. Hence, it can be assumed that these factors did not contribute to neurologic or histologic differences between groups. Loss of medium-sized neurons in the dorsolateral caudate nucleus has been a consistent finding following global cerebral ischemia, although the magnitude of neuronal damage varies depending on some experimental variables, mainly the species and the duration of ischemia (19–22,29,35). In the present study, a loss of 43% of the population of mediumsized caudate neurons was observed following 17–19 min of acute global cerebral ischemia. An imbalance between excitatory and inhibitory neurotransmission has been proposed as a main factor leading to neuronal damage in the striatum following a period of cerebral ischemia. In particular, abnormally augmented glutamatergic and dopaminergic excitatory activity has been shown to be involved in neuronal damage affecting mediumsized neurons in the dorsolateral striatum, including an important proportion of GABAergic neurons (26–31).

Figure 2. Representative photomicrographs of the neuronal population found in the substantia nigra pars reticularis of cats subjected to sham procedures (SHAM), and cats subjected to acute global cerebral ischemia under either vehicle (ISCH  VEH), or progesterone (ISCH  P4) treatment. No significant differences were found in the densities of neurons among cats under the various experimental conditions. Luxol fast blue and cresyl violet. Scale bar, 100 m.

A consequence of the lesion of medium-sized neurons of the caudate nucleus is the reduction of the neuronal population of the SNr, as observed in other experimental models in which single or repetitive episodes of global cerebral ischemia were induced (57–59). This has been interpreted as due to transneuronal degeneration of neurons in the SNr resulting from the lack of normal GABAergic innervation from the medium-sized GABAergic neurons of the caudate nucleus and the globus pallidus projecting to the reticulatatype neurons of the SNr and the lateral SN leading to an imbalance between excitatory and inhibitory inputs, thus enhancing glutamate-mediated excitotoxicity (40,41,57–59).

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Table 4. Number of large (1542 m longest diameter) and small (1015 m diameter) neurons found in microscopy fields of 450 m of diameter in the substantia nigra pars reticularis of cats under the various experimental conditions Number of large neurons/field (Md, range) Sham Ischemia  vehicle Ischemia  P4

8.3 7.8 5.8

3.58.6 2.710.1 0.511.3

Number of small neurons/field (Md, range) 2.9 3.5 3.6

0.75.1 1.45.0 1.08.2

It is known that neurotoxic damage in the striatum destroys 95% of caudate neurons resulting in secondary neuronal damage in the SNr (56), and that extensive damage of the striatum is a necessary condition for transneuronal degeneration of SNr neurons following ischemia (57). Nonetheless, in this case detailed quantitative data concerning the magnitude of striatal damage in terms of its neuronal population and the resulting loss of SNr neurons have not been described. In the present study it appears that the severity of the caudate damage, with 43% loss of medium-sized neurons, was not of sufficient magnitude to produce a secondary, significant reduction of SNr neuronal population 14 days after ischemia. Present results support the neuroprotective effect of P4 on the medium-sized neurons of the dorsolateral caudate nucleus. The possibility exists that some of the effects found may be mediated by P4 biotransformation to some of its 3,5- and 3,5 -reduced neuroactive metabolites, as suggested for other effects of P4 on the brain (75–77). In fact, two of the key steroid-metabolizing enzymes, 5-reductase and 3-hydroxysteroid oxidoreductase, are widely distributed in the brain (78) and although lower than in other brain areas, their activity has been demonstrated both in the hippocampus and in the striatum (79). A neuroprotective effect of P4 treatment as shown by preservation of pyramidal neurons of the hippocampus and better neurologic outcome following acute global ischemia in cats (53) has been explained as due to an enhancement of GABAergic activity in the central nervous system. It has been demonstrated that the neuroactive metabolites of P4 may interact with the GABAA receptor, increasing the inward Cl currents at both presynaptic and postsynaptic levels (60–65). Thus, inhibition of neuronal excitability (75) and a reduction in the release of excitatory neurotransmitters (80) may account for the neuroprotective effect of these steroids under different cerebral injury conditions (81–88). A GABAergic-mediated neuroprotective mechanism could also be involved in the preservation of the medium-sized caudate neurons in the present study under P4 treatment because these neurons receive GABAergic innervation, thus rendering them suitable targets for neuroprotective drugs enhancing GABAergic neurotransmission. In fact, both direct and indirect GABA agonists exert a neuroprotective ef-

fect on caudate and SNr neurons following forebrain ischemia in gerbils and rats (40,41,44–46). In addition, enhancement of GABAergic inhibitory activity induced by P4 treatment may counteract the ischemia-induced excitotoxic phenomena associated with excessive glutamatergic and dopaminergic activity, thus resulting in neuroprotection of the vulnerable neurons of the caudate nucleus. Other mechanisms in addition to the increase in GABAergic activity may contribute to the neuroprotective effects of progesterone on the caudate nucleus neuronal loss observed in the present study. Progesterone has been shown to attenuate lipid peroxidation induced by FeSO4 and amyloid -peptide, protect neuronal cultures against glutamate toxicity and glucose deprivation (89), and reduce lipid peroxidation after cortical contusion in rats (83). Because an oxidative damage has been proposed to contribute to striatal damage after ischemia (29–31), a possible reduction of lipid peroxidation by P4 may contribute to its neuroprotective effects found in the present study. Further studies should be undertaken to confirm these possibilities. Overall data support the conclusion that the neuroprotective effects of P4 are not limited to the highly vulnerable pyramidal neurons of the hippocampus but may also be exerted in the neuronal components of other brain structures such as the caudate nucleus. This idea is consistent with the possibility that alterations in neurologic phenomena whose integration depends on the functioning of these brain structures, aside from the hippocampus, may also be reduced under progesterone treatment. Acknowledgments This work was partially supported by a research grant from the Consejo Nacional de Ciencia y Tecnología (CONACYT 3400PM0896), Mexico.

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