Increased axonal regrowth of lesioned rat sciatic nerve by veratrylguanidine methane sulfonate.

Neurotrophic factors appear as essential factors for normal development and repair of the nervous tissue. Veratrylguanidine methane sulfonate, has been shown to induce important neurite outgrowth of cultured dorsal root ganglia isolated from newborn rats. Its action was similar to that of NGF and was found to be additive to that of NGF. In order to see if this compound was able to stimulate axonal growth in adult animals, we examined the effect of this substance on the regeneration of the lesioned sciatic nerve. Using histochemical, immunohistochemical and ultrastructural studies, it is shown that a single intraperitoneal injection of veratrylguanidine methane sulfonate significantly increases the axonal growth during repair of the adult rat sciatic nerve. The efficiency of this substance is explained by its good targeting and long life time in the sciatic nerve.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

Possible involvement of A1 receptors in the inhibition of gonadotropin secretion induced by adenosine in rat hemipituitaries in vitro

We investigated the participation of A1 or A2 receptors in the gonadotrope and their role in the regulation of LH and FSH secretion in adult rat hemipituitary preparations, using adenosine analogues. A dose-dependent inhibition of LH and FSH secretion was observed after the administration of graded doses of the R-isomer of phenylisopropyladenosine (R-PIA; 1 nM, 10 nM, 100 nM, 1 µM and 10 µM). The effect of R-PIA (10 nM) was blocked by the addition of 8-cyclopentyltheophylline (CPT), a selective A1 adenosine receptor antagonist, at the dose of 1 µM. The addition of an A2 receptor-specific agonist, 5-N-methylcarboxamidoadenosine (MECA), at the doses of 1 nM to 1 µM had no significant effect on LH or FSH secretion, suggesting the absence of this receptor subtype in the gonadotrope. However, a sharp inhibition of the basal secretion of these gonadotropins was observed after the administration of 10 µM MECA. This effect mimicked the inhibition induced by R-PIA, supporting the hypothesis of the presence of A1 receptors in the gonadotrope. R-PIA (1 nM to 1 µM) also inhibited the secretion of LH and FSH induced by phospholipase C (0.5 IU/ml) in a dose-dependent manner. These results suggest the presence of A1 receptors and the absence of A2 receptors in the gonadotrope. It is possible that the inhibition of LH and FSH secretion resulting from the activation of A1 receptors may have occurred independently of the increase in membrane phosphoinositide synthesis.

Développement physiologique des voies visuelles chez le rat normal et chez celui ayant subi des convulsions hyperthermiques

Les neurones des couches superficielles du collicule supérieur et du cortex visuel primaire du rat adulte sont sensibles à de basses fréquences spatiales de haut contraste défilant à des vitesses élevées. Entre les jours post-nataux 27-30 et l’âge adulte, les fréquences temporelles optimales des neurones du cortex visuel primaire augmentent, tandis que leurs seuils de contraste diminuent. Cependant, les fréquences spatiales optimales, les valeurs de résolution spatiale et les bandes passantes spatiales de ces neurones sont, dès l’ouverture des paupières, similaires à celles observées chez le rat adulte. Ces profils de réponse neuronale suggèrent que les projections rétino-colliculaires et rétino-géniculo-corticales sont essentiellement issues de neurones ganglionnaires rétinofuges magnocellulaires et koniocellulaires. Les neurones du cortex visuel primaire du rat ayant subi des convulsions hyperthermiques présentent, dès l’ouverture des paupières, de basses fréquences spatiales optimales, de larges bandes passantes directionnelles et temporelles ainsi que des seuils de contraste élevés par rapport aux neurones du rat normal. À l’âge adulte, de basses fréquences temporelles optimales et de larges bandes passantes spatiales sont également observées chez le rat ayant subi des convulsions hyperthermiques. L’altération des profils de réponse des neurones du cortex visuel primaire du rat ayant subi de convulsions hyperthermiques suggère un déséquilibre entre les mécanismes neuronaux excitateurs et inhibiteurs de cette aire corticale. Ces résultats suggèrent également qu’un épisode unique de convulsions fébriles infantiles suffit à altérer le développement des propriétés spatio-temporelles des champs récepteurs des neurones du cortex visuel primaire.

Over expression of Plk1 does not induce cell division in rat cardiac myocytes in vitro

BACKGROUND: Mammalian cardiac myocytes withdraw from the cell cycle during post-natal development, resulting in a non-proliferating, fully differentiated adult phenotype that is unable to repair damage to the myocardium, such as occurs following a myocardial infarction. We and others previously have shown that forced expression of certain cell cycle molecules in adult cardiac myocytes can promote cell cycle progression and division in these cells. The mitotic serine/threonine kinase, Polo-like kinase-1 (Plk1), is known to phosphorylate and activate a number of mitotic targets, including Cdc2/Cyclin B1, and to promote cell division. PRINCIPAL FINDINGS: The mammalian Plk family are all differentially regulated during the development of rat cardiac myocytes, with Plk1 showing the most dramatic decrease in both mRNA, protein and activity in the adult. We determined the potential of Plk1 to induce cell cycle progression and division in cultured rat cardiac myocytes. A persistent and progressive loss of Plk1 expression was observed during myocyte development that correlated with the withdrawal of adult rat cardiac myocytes from the cell cycle. Interestingly, when Plk1 was over-expressed in cardiac myocytes by adenovirus infection, it was not able to promote cell cycle progression, as determined by cell number and percent binucleation. CONCLUSIONS: We conclude that, in contrast to Cdc2/Cyclin B1 over-expression, the forced expression of Plk1 in adult cardiac myocytes is not sufficient to induce cell division and myocardial repair.

Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

EFFECTS of DIURON on MALE RAT REPRODUCTIVE ORGANS: A DEVELOPMENTAL and POSTNATAL STUDY

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Long-term effects of developmental exposure to di-n-butyl-phthalate (DBP) on rat prostate: Proliferative and inflammatory disorders and a possible role of androgens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars

Background and Objectives: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis.Methods: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy.Results: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles - images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez.Conclusions: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP > AP > DP > VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma. © 2006 International Federation for Cell Biology.

Possible involvement of A1 receptors in the inhibition of gonadotropin secretion induced by adenosine in rat hemipituitaries in vitro

We investigated the participation of A₁ or A₂ receptors in the gonadotrope and their role in the regulation of LH and FSH secretion in adult rat hemipituitary preparations, using adenosine analogues. A dose-dependent inhibition of LH and FSH secretion was observed after the administration of graded doses of the R-isomer of phenylisopropyladenosine (R-PIA; 1 nM, 10 nM, 100 nM, 1 µM and 10 µM). The effect of R-PIA (10 nM) was blocked by the addition of 8-cyclopentyltheophylline (CPT), a selective A₁ adenosine receptor antagonist, at the dose of 1 µM. The addition of an A₂ receptor-specific agonist, 5-N-methylcarboxamidoadenosine (MECA), at the doses of 1 nM to 1 µM had no significant effect on LH or FSH secretion, suggesting the absence of this receptor subtype in the gonadotrope. However, a sharp inhibition of the basal secretion of these gonadotropins was observed after the administration of 10 µM MECA. This effect mimicked the inhibition induced by R-PIA, supporting the hypothesis of the presence of A₁ receptors in the gonadotrope. R-PIA (1 nM to 1 µM) also inhibited the secretion of LH and FSH induced by phospholipase C (0.5 IU/ml) in a dose-dependent manner. These results suggest the presence of A₁ receptors and the absence of A₂ receptors in the gonadotrope. It is possible that the inhibition of LH and FSH secretion resulting from the activation of A₁ receptors may have occurred independently of the increase in membrane phosphoinositide synthesis.

Different protocols of physical exercise produce different effects on synaptic and structural proteins in motor areas of the rat brain

The plastic brain responses generated by the training with acrobatic exercise (AE) and with treadmill exercise (TE) may be different. We evaluated the protein expression of synapsin I (SYS), synaptophysin (SYP), microtubule-associated protein 2 (MAP2) and neurofilaments (NF) by immunohistochemistry and Western blotting in the motor cortex, striatum and cerebellum of rats subjected to TE and AE. Young adult male Wistar rats were divided into 3 groups: sedentary (Sed) (n=15), TE (n=20) and AE (n=20). The rats were trained 3 days/week for 4 weeks on a treadmill at 0.6 km/h, 40 min/day (TE), or moved through a circuit of obstacles 5 times/day (AE). The rats from the TE group exhibited a significant increase of SYS and SYP in the motor cortex, of NF68, SYS and SYP in the striatum, and of MAP2, NF and SYS in the cerebellum, whereas NF was decreased in the motor cortex and the molecular layer of the cerebellar cortex. On the other hand, the rats from the AE group showed a significant increase of MAP2 and SYP in the motor cortex, of all four proteins in the striatum, and of SYS in the cerebellum. In conclusion, AE induced changes in the expression of synaptic and structural proteins mainly in the motor cortex and striatum, which may underlie part of the learning of complex motor tasks. TE, on the other hand, promoted more robust changes of structural proteins in all three regions, especially in the cerebellum, which is involved in learned and automatic tasks. (C) 2012 Elsevier B.V. All rights reserved.