Physiopathology of ischemic stroke and its modulation using memantine: evidence from preclinical stroke.

Ischemic stroke is the most common type of cerebrovascular disease and is caused by an interruption of blood flow in the brain. In this disease, two different damage areas are identifying: the lesion core, in which cells quickly die; and the penumbra (surrounding the lesion core), in which cells are functionally weakened but may recover and restore their functions. The currently approved treatments for ischemic stroke are the recombinant tissue plasminogen activator and endovascular thrombectomy, but they have a short therapeutic window (4.5 and 6 hours after stroke onset, respectively) and a low percentage of stroke patients actually receive these treatments. Memantine is an approved drug for the treatment of Alzheimer's disease. Memantine is a noncompetitive, low affinity and use-dependent antagonist of N-methyl-D-aspartate glutamate receptor. Memantine has several advantages over developing a new drug to treat focal ischemic stroke, but the most important is that it has sufficient safe probes in preclinical models and humans, and if the preclinical studies provide more evidence about pharmacological actions in tissue protection and repair, this could help to increase the number of clinical trials. The present review summarizes the physiopathology of isquemic stroke and the pharmacological actions in neuroprotection and neuroplasticity of memantine in the post stroke stage of preclinical stroke models, to illustrate their potential to improve functional recovery in human patients.

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

The content of gonadotropin-releasing hormone (GnRH), kisspeptin, and estrogen receptors (ERα/ERß) in the anteromedial hypothalamus displays daily variations throughout the rat estrous cycle.

The content of gonadotropin-releasing hormone (GnRH), its mRNA, and estrogen receptor alpha (ERα) and beta (ERß) in the hypothalamus varies throughout the estrous cycle. Furthermore, the abundance of these molecules displays asymmetry between the right and left side. In the present study, we investigated the changes in the content of ERα, ERß, kisspeptin, and GnRH by western blot in the left and right anteromedial hypothalamus, at four different times during each stage of the rat estrous cycle. The serum levels of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were also measured. ERα and ERß levels changed depending on the stage of the estrous cycle, meanwhile that of kisspeptin was modified according to both the hour of the day and the stage of the cycle. Except in estrus day, ERß was higher in the right hypothalamus, while ERα was similar in both sides. During both proestrus and estrus, the content of kisspeptin and GnRH was higher in the right hypothalamus. The highest levels of FSH and LH occurred at 17:00 h of proestrus. But at estrus, the highest FSH levels were observed at 08:00 h and the lowest at 17:00 h. Thus, the current results show that the content of ERα, ERß, kisspeptin, and GnRH in the anteromedial hypothalamus are regulated as a function of the stage of the estrous cycle and the hour of the day. Furthermore, the content of these proteins is regularly higher in the right anteromedial hypothalamus, regardless of the stage of the cycle or time of the day.

Estrogen Receptors Alpha and Beta in POA-AHA Region Regulate Asymmetrically Ovulation.

We examined the role of the estrogen receptors alpha (ERα) and beta (ERß) in of the preoptic-anterior hypothalamic area (POA-AHA) in the regulation of ovulation in rats. The number of ERα- and ERß-immunoreactive (-ir) cells was determined at 09:00, 13:00, and 17:00 h of each stage of the estrous cycle in intact rats. Additionally, the effects of blocking ERα and ERß on ovulation rate at 09:00 h on diestrus-2 or proestrus day through the microinjection of methyl-piperidino-pyrazole (MPP) or cyclofenil in either side of POA-AHA were evaluated. The number of ERα-ir and ERß-ir cells in POA-AHA varied in each phase of estrous cycle. Either MPP or cyclofenil in the right side of POA-AHA on diestrus-2 day reduced the ovulation rate, while at proestrus day it was decreased in rats treated in either side with MPP, and in those treated with cyclofenil in the left side. MPP or cyclofenil produced a decrease in the surge of luteinizing hormone levels (LH) and an increase in progesterone and follicle stimulating hormone (FSH). Replacement with synthetic luteinizing hormone-releasing hormone in non-ovulating rats treated with MPP or cyclofenil restored ovulation. These results suggest that activation of estrogen receptors on the morning of diestrus-2 and proestrus day asymmetrically regulates ovulation and appropriately regulates the secretion of FSH and progesterone in the morning and afternoon of proestrus day. This ensures that both, the preovulatory secretion of LH and ovulation, occur at the right time.

Muscarinic Receptors Types 1 and 2 in the Preoptic-Anterior Hypothalamic Areas Regulate Ovulation Unequally in the Rat Oestrous Cycle.

Muscarinic receptors types 1 (m1AChR) and 2 (m2AChR) in the preoptic and anterior hypothalamus areas (POA-AHA) were counted, and the effects of blocking these receptors on spontaneous ovulation were analysed throughout the rat oestrous cycle. Rats in each phase of the oestrous cycle were assigned to the following experiments: (1) an immunohistochemical study of the number of cells expressing m1AChR or m2AChR in the POA-AHA and (2) analysis of the effects of the unilateral blockade of the m1AChR (pirenzepine, PZP) or m2AChR (methoctramine, MTC) on either side of the POA-AHA on the ovulation rate. The number of m2AChR-immunoreactive cells was significantly higher at 09:00 h on each day of the oestrous cycle in the POA-AHA region, while no changes in the expression profile of m1AChR protein were observed. The ovulation rate in rats treated with PZP on the oestrous day was lower than that in the vehicle group. Animals treated on dioestrous-1 with PZP or MTC had a higher ovulation rate than those in the vehicle group. In contrast, on dioestrous-2, the MTC treatment decreased the ovulation rate. These results suggest that m1AChR or m2AChR in the POA-AHA could participate in the regulation of spontaneous ovulation in rats.

The participation of the muscarinic receptors in the preoptic-anterior hypothalamic areas in the regulation of ovulation depends on the ovary.

BACKGROUND: Muscarinic receptors (mAChRs) of the preoptic and anterior hypothalamus areas (POA-AHA) regulate ovulation in an asymmetric manner during the estrous cycle. The aims of the present study were to analyze the effects of a temporal blockade of mAChRs on either side of the POA-AHA performed in diestrus-2 rats on ovulation, the levels of estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) and the mechanisms involved in changes in ovulation. METHODS: Cyclic rats on diestrus-2 day were anesthetized and randomly assigned to the following groups: 1) microinjection of 1 µl of saline or atropine solution (62.5 ng) in the left or right POA-AHA; 2) removal (unilateral ovariectomty, ULO) of the left (L-ULO) or right (R-ULO) ovary, and 3) rats microinjected with atropine into the left or right POA-AHA plus L-ULO or R-ULO. The ovulation rate and the number of ova shed were measured during the predicted estrus, as well as the levels of estradiol, FSH and LH during the predicted proestrus and the effects of injecting synthetic LH-releasing hormone (LHRH) or estradiol benzoate (EB). RESULTS: Atropine in the left POA-AHA decreased both the ovulation rate and estradiol and LH levels on the afternoon of proestrus, also LHRH or EB injection restored ovulation. L- or R-ULO resulted in a lower ovulation rate and smaller number of ova shed, and only injection of LHRH restored ovulation. EB injection at diestrus-2 restored ovulation in animals with L-ULO only. The levels of estradiol, FSH and LH in rats with L-ULO were higher than in animals with unilateral laparotomy. In the group microinjected with atropine in the left POA-AHA, ovulation was similar to that in ULO rats. In contrast, atropine in the right POA-AHA of ULO rats blocked ovulation, an action that was restored by either LHRH or EB injection. CONCLUSIONS: These results indicated that the removal of a single ovary at noon on diestrus-2 day perturbed the neuronal pathways regulating LH secretion, which was mediated by the muscarinic system connecting the right POA-AHA and the ovaries.

Effects of unilaterally microinjecting ethanol in the preoptic-anterior hypothalamic areas of rats on ovulation.

BACKGROUND: Intragastric or intraperitoneal ethanol (EtOH) treatment inhibits reproductive functions in females and male rats. The area of the hypothalamus where these effects take place is unknown. As the participations of the preoptic-anterior hypothalamic area (POA-AHA) in regulating ovulation is asymmetric, this study aims to analyze the effects on 17ß-estradiol(E2 ), progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) serum levels, the messenger ribonucleic acid (mRNA) expression of estrogen receptor alpha (ERα) and beta (ERß), and ovulation resulting from unilaterally microinjecting water or an EtOH solution into either side of the POA-AHA. METHODS: The treatment consisted of microinjecting a 8.6 µM EtOH solution into either side of the POA-AHA. The study was performed on groups of adult cyclic rats at 09.00 hours on diestrus-1, and sacrificed on diestrus-2 at 13.00, on proestrus at 09.00 or 17.00 or on estrus at 09.00 hours. Ovulation rates were assessed in rats sacrificed on estrus. Hormonal serum levels were measured using radioimmunoassay, and as a function of ERα and ERß mRNA expression in each side of the POA-AHA by reverse transcriptase polymerase chain reaction. RESULTS: EtOH treatment blocked ovulation and the preovulatory release of LH, and lowered E2 levels. Irrespective of the treated POA-AHA side, ERα mRNA expression was consistently lower in the left POA-AHA and higher on the right. EtOH treatment in the left POA-AHA decreased FSH serum levels and lowered ERß mRNA expression. In turn, EtOH treatment on the right POA-AHA resulted in higher FSH levels and ERß mRNA expression. CONCLUSIONS: The present results show that EtOH blocks the preovulatory surge of LH on the POA-AHA. The effects of EtOH treatment of preovulatory FSH surge on the POA-AHA are asymmetric (stimulative on the right and inhibiting in the left). The effects of EtOH treatment on preovulatory LH and FSH surge could be explained by the inhibition of ERα and ERß mRNA expression, respectively.

The cholinergic system of the preoptic-anterior hypothalamic areas regulates the ovarian follicular population in an asymmetric way.

Atropine implants in the preoptic-anterior hypothalamic areas (POA-AHA) block ovulation. The blocking effects depend on the side of POA-AHA and the day of the estrous cycle in which the implants are inserted. Since ovulation is the result of the growth and differentiation of ovarian follicles, the purpose of this study was to analyze the changes in follicular and atresia population in the ovaries of non-ovulating rats resulting from the unilateral atropine implants in the POA-AHA. Groups of cyclic rats were implanted with atropine or cholesterol (sham treatment group) in the left (diestrus-1, diestrus-2) or the right side (estrus, diestrus-1) of the POA-AHA. The animals were sacrificed on the expected proestrus or estrus day, and the follicular population was counted and the follicles measured in both ovaries. Atropine implants inserted in the left POA-AHA on diestrus-2 resulted in lower follicular growth and atresia in the ipsilateral ovary (left one). No apparent effects were observed in the right ovary. Atropine implants inserted in the right POA-AHA on estrus day resulted in fewer numbers of small follicles in the ipsilateral ovary (right) and a greater number of pre-ovulatory ones. Present results suggest that acetylcholine, via muscarinic receptors of the POA-AHA, regulates ovarian follicular fate in an asymmetric way, and that its actions fluctuate during the estrous cycle. In addition, each ovary seems to respond differently to the POA-AHA's muscarinic signal surge on estrus and diestrus-2 days.

Acute effects of unilateral sectioning the superior ovarian nerve of rats with unilateral ovariectomy on ovarian hormones (progesterone, testosterone and estradiol) levels vary during the estrous cycle.

The present study analyzed the participation of the left and right superior ovarian nerves (SON) in regulating progesterone, testosterone, and estradiol serum levels in unilaterally ovariectomized rats on each day of the estrous cycle. For this purpose, ovarian hormone concentrations in serum were measured in animals with either sham-surgery, unilateral ovariectomy (ULO), unilateral sectioning of the SON, or sectioning of the SON innervation of the in situ ovary in rats with ULO.This investigation results show that the right and left ovaries have different capacities to maintain normal hormone levels, that such capacity varies during the estrous cycle, and that it depends on the integrity of the SON innervation. In rats with only one ovary, the effects of ovarian denervation on hormone levels varied according to which ovary remained in situ, the specific hormone, and the day of the estrous cycle when treatment was performed. Present results support the idea that the ovaries send and receive neural information that is processed in the central nervous system and we propose that this information participates in controlling the secretion of gonadotropins related to the regulation of ovarian functions.

Estandarización de la Figura de Taylor en población mexicana / Taylor's Figure Standardization on Mexican population

In order to perform a neuropsychologycal evaluation, the clinician may use several instruments; nevertheless, most of them have been designed for use on populations with very different social and cultural backgrounds from that of Mexico. This makes the research on the standardization of methods of evaluation for Mexican population a very important task for both clinical and research settings. Normative data obtained from Mexican population is necessary because it provides the clinician that works with Mexican patients with a reference framework that allows him or her to correctly classify a particular behavior of an individual as normal or abnormal and thus make specific evaluations and cooperate in diagnostic. Researchers interested on cognitive functioning also require qualitative and quantitative equivalent instruments that may allow them to objectively evaluate the efficacy of short-time interventions as in a pre- and post-treatment experimental designs; and it is precisely for this reason that Taylor's figure was developed. Taylor's figure (TF) was originally designed as an alternative to Rey-Osterrieth's complex figure (ROCF), in order to use it in test-retest situations. Similar to ROCF, Taylor's figure has two modalities: copy and memory. The former evaluates constructional praxia, while the latter measures immediate recalling. Parallel tests, that is, different tests that evaluate the same variables, are useful because they reduce the measurement error involved in applying the task twice to the same person (i.e. learning), thus increasing the validity of follow up evaluations of cognitive functioning. <

Dentro del ámbito neuropsicológico existe una serie de instrumentos de evaluación que en su mayoría han sido diseñados en un contexto sociocultural diferente al nuestro, por lo que es importante contar con pruebas neuropsicológicas estandarizadas en sujetos mexicanos. Desde esta perspectiva, los datos de una población normativa nos permiten tener un marco de referencia para comparar y establecer diagnósticos diferenciales dentro de la práctica clínica. Asimismo, dentro del ámbito de la investigación se requieren instrumentos que en teoría sean equivalentes en cuanto a la función que evalúan para llevar un seguimiento sobre el funcionamiento cognoscitivo de una población en particular a lo largo del tiempo. La Figura de Taylor se diseñó como una alternativa a la Figura Compleja de Rey-Osterrieth para aplicarla en situaciones de test-retest. Consta de dos modalidades: una de copia que evalúa la praxia de construcción y otra de memoria inmediata. El término <

The acute effects of bilateral ovariectomy or adrenalectomy on progesterone, testosterone and estradiol serum levels depend on the surgical approach and the day of the estrous cycle when they are performed.

Bilateral ovariectomy or adrenalectomy are experimental tools used to understand the mechanisms regulating the hypothalamus-pituitary-ovarian and the hypothalamus-pituitary-adrenal axis. There is evidence that acute unilateral perforation of the dorsal peritoneum in rats results in significant changes in progesterone, testosterone and estradiol serum concentrations. Because different surgical approaches for unilateral or bilateral ovariectomy or adrenalectomy, sectioning the superior ovarian nerve or the vagus nerve are used, we compare the acute effects on hormone serum concentrations resulting from the unilateral or bilateral dorsal approach to performing bilateral ovariectomy or adrenalectomy with those obtained when an unilateral incision is performed in the ventral abdomen. In general, the progesterone, testosterone and estradiol serum concentrations were higher in animals with ventral approach than in those with dorsal surgery, the effects varying depending on the day of the estrous cycle when surgery was performed. The results suggest that the neural signals arising from different zones of the peritoneum and/or the abdominal wall play different roles in the mechanisms regulating steroid hormones concentrations.

Asymmetric effects of acute hemiovariectomy on steroid hormone secretion by the in situ ovary.

The acute effects of hemiovariectomy on progesterone, testosterone, estradiol, and luteinizing hormone (LH) concentrations in serum were studied in rats under the following experimental conditions: control, shamoperated (left or right), hemiovariectomized, bilateral adrenalectomized, and hemiovariectomized plus bilateral adrenalectomized. One-hour after surgery, the concentration of progesterone and testosterone in the serum of right-side sham-operated rats was significantly higher than in control animals. Testosterone concentration in serum in rats with the right ovary in situ was higher than in sham-operated animals; injecting atropine sulfate 1 h before surgery blocked such increase, while the same treatment to rats with the left ovary remaining in situ resulted in a significant increase of testosterone concentration. Adrenalectomy resulted in an increase of testosterone concentration, which was higher when atropine sulfate was injected before surgery. Our results support the idea that left and right ovaries play different roles in the regulation of hormone secretion, and that such differences are related to ovarian innervation.

Differential mRNA expression of alpha and beta estrogen receptor isoforms and GnRH in the left and right side of the preoptic and anterior hypothalamic area during the estrous cycle of the rat.

Asymmetric mRNA expression was found in preoptic anterior and hypothalamic anterior areas of the two estrogen receptor isoforms and the gonadotropin-releasing hormone. On the right side of these areas, estrogen receptor alpha mRNA expression reached its peak on estrus day, while on the left side the peak was reached on proestrus day. Estrogen receptor beta mRNA expression peaked on both sides on the same day, diestrous-2 day, but at different hours, showing a sustained expression for the next measured hour on the left side, while peaking and dropping abruptly on the right side. Gonadotropin-releasing hormone also peaked on both sides on diestrous-2 day, being the left side peak expression significantly lower than the peak expression at the right side. The side expression differences suggest that different sides of the before mentioned areas may play different roles of endocrine reproductive functions, while differences of expression at different times may suggest interaction between sides for the same functions.

Expression of p53 in luminal and glandular epithelium during the growth and regression of rat uterus during the estrous cycle.

It has been well recognized that epithelial cells of the rat endometrium cyclically proliferate and die during the estrous cycle. The aim of the present study was to determine p53 expression pattern and correlate it with the the apoptotic pattern of epithelial cells of the rat uterus during the estrous cycle. The p53 mRNA and protein expression pattern was assessed by in situ hybridization and immunohistochemistry. The apoptotic index was determined by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and electron microscopy. The highest p53 mRNA content, detected by in situ hybridization, was observed on the metestrus day both in the luminal and the glandular epithelia. During this period both epithelia presented high proliferation. The content of p53 mRNA markedly decreased in the following days, presenting its minimal values on the estrus day. The highest number of p53 immunopositive nuclei, in both the luminal and the glandular epithelia, was also detected on the metestrus day, while the lowest one was found on estrus day. On the proestrus day, p53 protein was predominantly detected in the glandular epithelium. However, on the estrus day, p53 protein was detected both in the nuclei and in the cytoplasm of luminal epithelial cells, predominantly in the cytoplasm. The highest apoptotic index in both the luminal and the glandular epithelia was observed on the estrus day whereas the lowest one was observed on the proestrus day. The apoptotic index values were higher in the luminal than in the glandular epithelia. The overall results indicate that p53 expression at both mRNA and protein levels is higher on the metestrus day when the apoptotic index is low. This suggests that p53 should play an important physiological role during proliferative phases of the estrous cycle in the rat uterus.

Asymmetrical effects of the unilateral implant of pilocarpine on the preoptic-anterior hypothalamic area on spontaneous ovulation of the adult rat

The effects on ovulation at the next strus after unilaterally implanting pilocarpine in the preoptic-anterior hypothalamic area (POA-AHA) of rats on each day of the estrous cycle were analyzed. Implantation on the left side of POA-AHA on the day of estrus blocked ovulation in all animals, whereas implantation on the right side did not (0/5 vs. 4/4, p<0.05). Implantation on diestrus 1 or 2 on either side of the POA-AHA blocked ovulation. Implatation on the righ side of the POA-AHA at the day of proestrus blocked ovulation (1/6 animals ovulated), while 10/12 with pilocarpine on the left side ovulated (p<0.05).The administration of 3.7 µg of GnRH at 13:00 h o the expected day of proestrus induced ovulation in 36/42 treated animals. In rat with a pilocarpine implant, the injection of estradiol benoznate on diestrus 2 restored ovulation only in those animals with the pilocarpine implant placed in the left side of the POA-AHA, performed on the day of estrus. The results support the previous estatements that in the adult rat POA-AHA, the cholinergic mechanism regulating preovulatory GnRH release, is lateralized. In addition, at the beginning of the estrous phase, the PAO-AHA-cholinergic system needs to remain undisturbed for normal ovulation to take place at the next estrus

Modifications on the ovarian response to gonadotropins induced by catecholamine depletion in vagotomized adult rats

Con el fin de estudiar la participación de las catecolaminas (CA) en el proceso del crecimiento folicular y la ovulación en el animal adulto, se analizaron los efectos de la depleción aguda de CA por la administración de reserpina (RSP) (1 mg/KG) sobre la respuesta ovulatoria de animales tratados con FSH y LH. Dado que previamente se mostró que la sección bilateral de los nervios vago provoca aumento en el número de ovocitos liberados por animal ovulante se analizaron los efectos de la vagotomía bilateral previa a la denervación aguda provocada por RSP. La inyección de RSP 3 horas antes de la administración de FSH provocó aumento del número de ovocitos liberados (21.49 ñ 2.87 vs 11.84 ñ 1.58, P < 0.01), del peso de los ovarios (66.20 ñ 4.41 vs. 49.79 ñ 3.99 mg/100 peso corporal, P < 0.01) y disminución del número de folículos preovulatorios. En los animales tratados con RSP 3 horas antes que recibieran LH (53 horas después que fueron tratados con FSH) no se observaron cambios significativos en el número de ovocitos, ni en el peso de los ovarios, en relación con los testigos tratados con FSH y LH. La sección previa (20 días de los nervios vago bloqueó los efectos de la administración de RSP antes de la FSH (ovocitos 5.21 ñ 1.51 vs. 21.49 ñ 2.87, P < 0.001; peso de los ovarios 36.51 ñ 2.38 vs. 66.20 ñ 4.41 mg/100 g, P < 0.001). La sección bilateral de los nervios vago no modificó los resultados obtenidos por la administración de RSP antes de LH. Los resultados del presente estudio sugieren que en condiciones normales las CA tendrían efectos inhibidores sobre el número de receptores foliculares a FSH, mientras que no parecen intervenir en los receptores a LH vinculados al proceso de ovulación. El hecho que la sección bilateral de los nervios vago revierta los efectos de la inyección de RSP antes de la FSH sugiere que en condiciones normales la información que llevan los nervios vago al ovario es agonista con las CA