Progesterone treatment in rats after severe global cerebral ischemia promotes hippocampal dentate gyrus neurogenesis and functional recovery.

OBJECTIVE: Rats treated with progesterone (P4) after ischemia show an adequate functional performance despite a significant loss of hippocampal pyramidal neurons, suggesting that P4 could favour a permissive microenvironment for cerebral plasticity mechanisms. The possibility of P4 treatment promoting the survival of newly generated hippocampal neurons, in relation to the performance of ischemic rats in a spatial learning task, was assessed in this study. METHODS: Adult male rats were subjected to a severe global cerebral ischemia episode (30 min) and treated with P4 or its vehicle at 15 min, 2, 6, 24, 48 and 72 h of reperfusion. From day 4 to 8 post-ischemia 5-bromo-2-deoxyuridine (BrdU) was administered to label proliferating cells. Twenty-one days post-ischemia, the rats were exposed to the Morris water maze to assess behavioral parameters of spatial learning and memory. Subsequently, the brain was perfusion-fixed and immunofluorescence procedures were performed to quantify the number of new mature neurons (BrdU+/NeuN+) in the dentate gyrus (DG) of the hippocampus. RESULTS: Rats subjected to severe global cerebral ischemia and treated with P4 had a significantly better performance in spatial learning-memory tests, than those treated with vehicle, and a significantly higher number of new mature neurons (BrdU+/NeuN+) in the DG. CONCLUSION: These findings show that post-ischemia P4 treatment, following an episode of severe global cerebral ischemia, promotes the survival of newly generated hippocampal neurons in the DG, which may be one of the mechanisms of cerebral plasticity induced by the hormone, that underlie a successful functional performance in learning and memory tests.

Effects of progesterone on neurite growth inhibitors in the hippocampus following global cerebral ischemia.

In this study, the effects of progesterone (P4) on the immunoreactivity to the neurite growth inhibitor Nogo-A, its receptor (Ng-R), and its effector Rho-A in the rat hippocampus, in association with parameters of spatial learning and memory following global cerebral ischemia, were assessed. Adult male rats were subjected to global cerebral ischemia (15 min), and treated with P4 or its vehicle at 15 min, 2, 6, 24, 48 and 72 h of reperfusion. Immunoreactivity to Nogo-A, Ng-R, and Rho-A was evaluated at 24 h, 72 h or 7 d, or at 14 d of reperfusion after rats were tested in the Morris Water Maze (MWM). Global cerebral ischemia induced an increase in Nogo-A, Ng-R, and Rho-A immunoreactivities in the cell bodies of CA1 pyramidal neurons at 24h after global cerebral ischemia, peaking at 72 h, and persisting 14 d later. In addition, at 72 h, a strong immunoreactivity was observed in the hippocampal layers where dendritic arborizations of CA1 pyramidal neurons are located. Treatment with P4 reduced Nogo-A, Ng-R, and Rho-A immunoreactivities in CA1, particularly at 72 h of reperfusion. These effects of P4 were consistent with the parameters of a more efficient spatial learning and memory in the MWM, as compared to vehicle-treated rats. Overall results suggest the reduction of neurite growth inhibitory molecules Nogo-A, Ng-R, and Rho-A, as a part of the restorative effects of progesterone possibly allowing the plastic phenomena to occur, able to support the functional preservation of the hippocampus following global cerebral ischemia.

Post-ischemic administration of progesterone reduces caspase-3 activation and DNA fragmentation in the hippocampus following global cerebral ischemia.

Delayed death of hippocampal CA1 pyramidal neurons following global cerebral ischemia/reperfusion may be mediated, in part, by caspase-3 activation resulting in DNA fragmentation. Progesterone (P4) is known to exert neuroprotective effects in several models of brain injury. This study was designed to assess the effect of P4 on caspase-3 levels and activation, and DNA fragmentation in the hippocampus following global cerebral ischemia/reperfusion. Adult male Sprague-Dawley rats were subjected to global ischemia by the four-vessel occlusion model. P4 (8 mg/kg), or its vehicle were administered i.v. at 15 min, 2, 6, 24, 48 and 70 h of reperfusion. Remaining pyramidal neurons were assesed by the Nissl staining technique, caspase-3 levels and activation by immunohistochemistry and an in situ activity assay, and DNA fragmentation by the TUNEL method. Post-ischemic progesterone treatment significantly reduced the ischemia/reperfusion-induced increase in caspase-3 levels and activation at 72 h, and DNA fragmentation and CA1 neuronal loss at 7 days. Present results suggest the reduction of caspase-3 levels/activation, and DNA fragmentation, as a part of the neuroprotective effects of progesterone against global cerebral ischemia/reperfusion injury.

Cytoarchitectural characteristics of hippocampal CA1 pyramidal neurons of rats, four months after global cerebral ischemia and progesterone treatment.

PURPOSE: To analyze the cytoarchitectural characteristics of the remaining pyramidal neurons in the hippocampal CA1 subfield of rats, four months after global cerebral ischemia (GCI) and progesterone treatment. METHODS: Dendritic arborization, and density and shape of the dendritic spines of CA1 pyramidal neurons in brains of intact rats, or rats submitted 120 days earlier to GCI and treatment with progesterone (8 mg/kg) or its vehicle, at 15 min, and 2, 6, 24, 48, and 72 h after the onset of reperfusion, were analyzed in samples processed by a modified Golgi method. RESULTS: Few impregnated CA1 pyramidal neurons were identified in the ischemic vehicle-treated rats, with a short apical dendrite devoid of bifurcations and dendritic spines. In contrast, the remaining CA1 pyramidal neurons sampled from ischemic progesterone-treated rats showed sinuously branched dendrites with similar number of bifurcations and whole density of spines, and higher proportional density of mushroom spines than those in the intact group. CONCLUSIONS: These cytoarchitectural characteristics may underlie the long-term preservation of place learning and memory functions seen after ischemia and progesterone neuroprotective treatment, possibly compensating for the severe reduction in neuronal population.

Neuroprotective effects of progesterone and allopregnanolone on long-term cognitive outcome after global cerebral ischemia.

PURPOSE: To assess the longterm neuroprotective effects of progesterone (P4) and allopregnanolone (ALLO) on functional and morphological parameters of the integrity of the hippocampus, after global cerebral ischemia. METHODS: Adult male Sprague-Dawley rats were subjected to a transient severe (20 min) forebrain ischemia (Isch) episode and treated with P4 or ALLO (8 mg/kg i.v.) or its vehicle, at 20 min, 2, 6, 24, 48 and 72 h after ischemia. Rats subjected to Sham procedures, and intact rats were included as nonischemic controls. Three months after ischemia, both the functional (spatial learning and memory, and reference and working memory), and the morphological integrity (dimensions of the hippocampal formation, thickness of the CA1 subfield, and pyramidal neuron population) of the hippocampus and the medial prefrontal cortex(mPFC) were determined. RESULTS: Treatment with P4 or ALLO significantly reduced the impairment in spatial learning and memory, as well as in reference and working memory, and prevented the narrowing of the hippocampus, otherwise induced by ischemia. This better performance of P4 and ALLO treated rats than vehicle (Veh) treated rats, occurred in spite of a loss of pyramidal neurons in the CA1, CA2,CA3 and hilus subfields of the Ammon's horn (remaining neurons: Isch+Veh: 21.0, 35.6, 44.1, and 40.3%; Isch+P4: 19.9, 32.2,41.1, and 32.5%; Isch+ALLO: 25.5, 62.0, 73.7, and 56.7%), and nonsignificant changes in the mPFC, as compared to the Intact group (100%). CONCLUSIONS: Performance of P4 or ALLO treated rats in learning and memory tests suggests that these steroids promoted neural conditions accounting for adequate functioning long after ischemia, in spite of the loss of hippocampal pyramidal neurons.

Long-term evaluation of cytoarchitectonic characteristics of prefrontal cortex pyramidal neurons, following global cerebral ischemia and neuroprotective melatonin treatment, in rats.

Global cerebral ischemia induces alterations of working memory, as evidenced in the eight-arm radial maze, in the absence of significant changes of pyramidal neuron population in the prefrontal cortex. These alterations can be prevented by a neuroprotective melatonin treatment. Thus, the cytoarchitectonic characteristics of the pyramidal neurons located at layers III and V in the prefrontal cortex of rats that had been submitted 120 days earlier to acute global cerebral ischemia (15 min four-vessel occlusion), and melatonin (10 mg/(kgh) for 6h, i.v.) or vehicle administration, starting 30min after the end of cerebral blood flow interruption, were evaluated in order to gain information on the changes of the neural substrate underlying disruption of prefrontocortical functioning. Soma size, rough length and number of bifurcations of basilar and apical dendrites, as well as spine density and proportions of the different types of spines in a 50 microm length segment of a secondary dendrite branching from the apical and the basilar dendrites, of pyramidal neurons of the dorsal medial prefrontal cortex, were evaluated in Golgi material. A significant reduction of soma size, apical and basilar dendrite length, number of dendritic bifurcations, and spine density were observed in pyramidal neurons at layers III and V after cerebral ischemia, while these alterations were prevented by melatonin treatment. These cytoarchitectural differences between groups seem to underlie the observed alterations in spatial working memory of ischemic, vehicle-treated rats in the absence of pyramidal neuron loss, as well as the better display of these functions long after ischemia and melatonin neuroprotection.

Melatonin and ischemia-reperfusion injury of the brain.

This review summarizes the reports that have documented the neuroprotective effects of melatonin against ischemia/reperfusion brain injury. The studies were carried out on several species, using models of acute focal or global cerebral ischemia under different treatment schedules. The neuroprotective actions of melatonin were observed during critical evolving periods for cell processes of immediate or delayed neuronal death and brain injury, early after the ischemia/reperfusion episode. Late neural phenomena accounting either for brain damage or neuronal repair, plasticity and functional recovery taking place after ischemia/reperfusion have been rarely examined for the protective actions of melatonin. Special attention has been paid to the advantageous characteristics of melatonin as a neuroprotective drug: bioavailability into brain cells and cellular organelles targeted by morpho-functional derangement; effectiveness in exerting several neuroprotective actions, which can be amplified and prolonged by its metabolites, through direct and indirect antioxidant activity; prevention and reversal of mitochondrial malfunction, reducing inflammation, derangement of cytoskeleton organization, and pro-apoptotic cell signaling; lack of interference with thrombolytic and neuroprotective actions of other drugs; and an adequate safety profile. Thus, the immediate results of melatonin actions in reducing infarct volume, necrotic and apoptotic neuronal death, neurologic deficits, and in increasing the number of surviving neurons, may improve brain tissue preservation. The potential use of melatonin as a neuroprotective drug in clinical trials aimed to improve the outcome of patients suffering acute focal or global cerebral ischemia should be seriously considered.

Long-term study of dendritic spines from hippocampal CA1 pyramidal cells, after neuroprotective melatonin treatment following global cerebral ischemia in rats.

Melatonin reduces pyramidal neuronal death in the hippocampus and prevents the impairment of place learning and memory in the Morris water maze, otherwise occurring following global cerebral ischemia. The cytoarchitectonic characteristics of the hippocampal CA1 remaining pyramidal neurons in brains of rats submitted 120 days earlier to acute global cerebral ischemia (15-min four vessel occlusion, and melatonin 10mg/(kg h 6h), i.v. or vehicle administration) were compared to those of intact control rats in order to gain information concerning the neural substrate underlying preservation of hippocampal functioning. Hippocampi were processed according to a modification of the Golgi method. Dendritic bifurcations from pyramidal neurons in both the oriens-alveus and the striatum radiatum; as well as spine density and proportions of thin, stubby, mushroom-shaped, wide, ramified, and double spines in a 50 microm length segment of an oblique dendrite branching from the apical dendrite of the hippocampal CA1 remaining pyramidal neurons were evaluated. No impregnated CA1 pyramidal neurons were found in the ischemic-vehicle-treated rats. CA1 pyramidal neurons from ischemic-melatonin-treated rats showed stick-like and less ramified dendrites than those seen in intact control neurons. In addition, lesser density of spines, lower proportional density of thin spines, and higher proportional density of mushroom spines were counted in ischemic-melatonin-treated animals than those in the sinuously branched dendrites of the intact control group. These cytoarchitectural arrangements seem to be compatible with place learning and memory functions long after ischemia and melatonin neuroprotection.

Long-term morphological and functional evaluation of the neuroprotective effects of post-ischemic treatment with melatonin in rats.

Consensus on neuroprotection has pointed out the relevance of the long-term morphological and functional evaluation of the effectiveness of putative neuroprotective procedures. In the present study, place learning (Morris water maze) and working memory (eight-arm Olton radial maze) were evaluated in adult male rats 90 days after 15 min of global cerebral ischemia (four-vessel occlusion) followed by continuous i.v. infusion (10 mg/kg/hr) of melatonin (Isch + Mel) or vehicle (Isch + Veh) for 6 hr, and the pyramidal neuron population of the cornus Ammoni (CA) of the hippocampus and layers III and V of the medial prefrontal cortex was assessed at the end of the behavioral testing period (120 days after ischemia). Impairment of place learning, a significant delay in working memory acquisition, and a significant loss of pyramidal neurons in the Ammon's horn (CA1: 23%, CA2: 52% CA3: 73%, hilus: 64% remaining neurons), were observed in the Isch + Veh group. By contrast, a similar performance of the Isch + Mel group to that in the Intact and Sham groups and better than that of the Isch + Veh group, besides a significant reduction of pyramidal neuron loss in the CA subfields (CA1: 79%, CA2: 88% CA3: 86%, hilus: 72% remaining neurons), documented that melatonin treatment led to a long-term preservation of both the neural substrate, and the capability for integration of spatial learning and memory, mainly dependent on a normal hippocampal functioning. Overall the results emphasize the efficacy of melatonin in counteracting the pathophysiological processes induced by ischemia, by exerting its actions during a short but critical period early after the ischemic episode.

Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus.

Progesterone is neuroprotective in models of focal or global ischemia when treatment starts either before the insult or at the onset of reperfusion. In these cases the steroid may act during the occurrence of the early pathophysiological events triggered by ischemia or reperfusion. As opposed to this condition, the aim of the present study was to assess the effect of delayed, post-injury administration of progesterone on the preservation of pyramidal neurons of the hippocampus of rats 21 days after been exposed to global ischemia by the four vessel occlusion model. Progesterone (8 mg/kg, i.v.) or its vehicle, were administered at 20 min, 2, 6, and 24h after the end of ischemia. At histological examination, brains of the ischemic vehicle-treated rats showed a severe reduction of the population of pyramidal neurons in the CA1 and CA2 subfields (12% and 29% remaining neurons, respectively), and a less severe neuronal loss in the CA3 and CA4 subfields of the hippocampus (68% and 63% remaining neurons, respectively), as compared to rats exposed to sham procedures. They also showed a two-fold enlargement of the lateral ventricles and 33% shrinkage of the cerebral cortex as compared to the sham group. Progesterone treatment resulted in a significant preservation of pyramidal neurons in CA1 and CA2 (40% and 62% remaining neurons), with no ventricular dilation and only a mild (12%) cortical shrinkage. Results suggest that progesterone is able to interfere with some late pathophysiological mechanisms leading both to selective neuronal damage in the hippocampal CA1 and CA2 subfields, and to shrinkage of the cerebral cortex.

Detailed analysis of the male copulatory motor pattern in mammals: hormonal bases.

Data obtained, using a polygraphic technique, on the characteristics of the motor and genital copulatory responses of male rabbits, rats, mice, hamsters, and guinea pigs are reviewed. This methodology provided detailed information, not accessible to other analyses, on the frequency and dynamic organization of copulatory pelvic thrusting trains of the species studied. This comparative analysis showed that: (1) The male rat may display two types of ejaculatory responses, differing in the dynamic organization of the pelvic thrusting train, and in the duration of the intravaginal thrusting period preceding ejaculation. (2) In the guinea pigs and small rodents, but not in rabbits, pelvic thrusting at ejaculatory responses persists during intromission, and a period of fast intravaginal thrusting is associated with ejaculation. (3) The motor copulatory pattern of the rabbit, but not of the rat, hamster, or guinea pig, is affected by castration and hormone treatment, suggesting that, in rabbits, androgen acts both on motivation and on the spinal neural systems related to copulation.

Neuroprotective effects of progesterone on damage elicited by acute global cerebral ischemia in neurons of the caudate nucleus.

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 (P(4)) 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 P(4), were assessed in cats in the present study. METHODS: Ovariectomized adult cats were treated subcutaneously (s.c.) with either P(4) (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 P(4)-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 P(4) upon CN neuronal damage after ischemia.

Progesterone: Protective effects on the cat hippocampal neuronal damage due to acute global cerebral ischemia

Background. Barbiturates, benzodiazepines, and synthetic steroids having anesthetic properties, by enchacing the inhibitory GABAergic neurotransmission to the neruronal circuits of cerebral structures vulnerable to ischemia, reduce the damage induced by this condition. Some endogenous steroids resulting from progesterone (P4) biotransformation in the brain exert GABAaergic effects, thus inhibiting neuronal excitability. Hence, P4 administration both before and after an experimentally induced ischemic episode may prevent or decrease the ischemic cerebral damage. Methods. Ovariectomized adult cats were treated sc with either P4 (10 mg/kg/day) or corn oil during 7 days before and 7 days after being subjected to a period of acute global cerebral ischemia by 15 min of cardiorespiratory arrest followed by 4 min of reanimation. After 14 days of survival, animals were sacrificed and the brains perfused in situ and formalin-fixed for histological examination. Results. Acute global cerebral ischemia resulted in a severe loss of neurons (54-85 percent), mainly in CA1 and CA2 subfields of oil-treated cats. Progesterone significantly reduced the neuronal loss in those areas (21-49 percent). Conclusions. Overall results suggest that progesterone exerts protective effects against the neuronal cerebral damage induced by acute global cerebral ischemia

Effects of intrathecal administration of adrenergic agonists on the frequency of copulatory pelvic thrusting of the male rat

The effects of the intrathecal perispinal administration of adregergic agonists on the characteristic of frequency, duration, and vigor of pelvic thrusting displayed by male rats during copulation was assessed by an accelerometric technique. A different dose of one drug (noradrenaline, clonidine or isoproterenol) and saline as control was administered at the lumbosacral level of the spinal cord to sexually active male rats in tests of sexual behavior performed at weekly intervals. The intrathecal administration of noradrenaline (alpha-adrenoceptor agonist) increased the frequency of pelvic thrusting in mount and intromission responses, whereas both the alpha2-adrenoceptor agonist clinidine (25 µg) and the ß-adrenoceptor agonist isoproterenol ( 40 µg) reduced the frequency of pelvic thrusting in these responses as compared to values obtained under the intrathecal administration of saline. On the other hand, the duration of the thrusting trains and the potency or vigor of pelvic thrusting in mounts and intromissions did not differ from values obtained under saline treatment. These findings indicate a possible participation of noradrenaline in the modulation of the spinal mechanisms involved in the generation of rhytmic pelvic thrustint performed by the male rat during copulation