Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Anti-aversive effects of the atypical antipsychotic, aripiprazole, in animal models of anxiety

Aripiprazole is a unique antipsychotic that seems to act as a partial agonist at dopamine D2-receptors, contrasting with other drugs in this class, which are silent antagonists. Aripiprazole may also bind to serotonin receptors. Both neurotransmitters may play major roles in aversion-, anxiety-and panic-related behaviours. Thus, the present work tested the hypothesis that this antipsychotic could also have anti-aversive properties. Male Wistar rats received injections of aripiprazole (0.1-10 mg/kg) and were tested in the open field, in the elevated plus and T mazes (EPM and ETM, respectively) and in a contextual fear conditioning paradigm. Aripiprazole (1mg/kg) increased the percentage of entries onto the open arms of the EPM and attenuated escape responses in the ETM. In the latter model, the dose of 0.1 mg/kg also decreased the latency to leave the enclosed arm, suggesting anxiolytic- and panicolytic-like properties. This dose also decreased the time spent in freezing in a contextual fear conditioning. No significant motor effects were observed at these doses. The present data support the hypothesis that aripiprazole could inhibit anxiety-related responses. Acting as a partial agonist at dopamine receptors, this drug could effectively treat schizophrenia and, in contrast with most antipsychotic drugs, alleviate aversive states.

Anxiolytic-like effect of noradrenaline microinjection into the dorsal periaqueductal gray of rats

The brain noradrenergic system has been implicated in the expression of defensive behaviors elicited by acute stress. The dorsal periaqueductal gray area (dPAG) is a key structure involved in the behavioral and cardiovascular responses elicited by fear and anxiety situations. Although there are noradrenergic terminals in the dPAG, few studies have investigated the role of noradrenaline (NA) in the dPAG on anxiety modulation. The aim of this study was to evaluate the effect of NA microinjection into the dPAG of rats subjected to two animal models of anxiety, the elevated plus-maze and the Vogel conflict test. Male Wistar rats implanted with a guide cannula aimed at the dPAG received microinjections of NA (3, 15, or 45 nmol/0.05 mu l) or artificial cerebral spinal fluid into the dPAG immediately before being exposed to the elevated plus-maze or the Vogel conflict test. NA increased the exploration of the open arms and the number of enclosed arm entries in the elevated plus-maze. The increase in open arm exploration remained significant after being subjected to an analysis of covariance using the latter variable as covariate. Moreover, the NA microinjection into the dPAG did not increase general exploratory activity of animals subjected to the open-field test, indicating that the increase in open arm exploration cannot be attributed to a nonspecific increase in exploratory activity. In the Vogel test, the NA microinjection into the dPAG increased the number of punished licks without changing the number of nonpunished licks or interfering with the tail-flick test. The results, therefore, indicate that the NA microinjection into the dPAG produces anxiolytic-like effects, suggesting its possible involvement in the anxiety modulation. Behavioural Pharmacology 20:252-259 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

The expression of contextual fear conditioning involves activation of an NMDA receptor-nitric oxide pathway in the medial prefrontal cortex

The ventral portion of medial prefrontal cortex (vMPFC) is involved in contextual fear-conditioning expression in rats. In the present study, we investigated the role of local N-methyl-D-aspartic acid (NMDA) glutamate receptors and nitric oxide (NO) in vMPFC on the behavioral (freezing) and cardiovascular (increase of arterial pressure and heart rate) responses of rats exposed to a context fear conditioning. The results showed that both freezing and cardiovascular responses to contextual fear conditioning were reduced by bilateral administration of NMDA receptor antagonist LY235959 (4 nmol/200 nL) into the vMPFC before reexposition to conditioned chamber. Bilateral inhibition of neuronal NO synthase (nNOS) by local vMPFC administration of the N omega-propyl-L-arginine (N-propyl, 0.04 nmol/200 nL) or the NO scavenger carboxy-PTI0 (1 nmol/200 A) caused similar results, inhibiting the fear responses. We also investigated the effects of inhibiting glutamate- and NO-mediated neurotransmission in the vMPFC at the time of aversive context exposure on reexposure to the same context. It was observed that the 1st exposure results in a significant attenuation of the fear responses on reexposure in vehicle-treated animals, which was not modified by the drugs. The present results suggest that a vMPFC NMDA-NO pathway may play an important role on expression of contextual fear conditioning.

Anxiolytic-like effects induced by acute reversible inactivation of the bed nucleus of stria terminalis

There is conflicting evidence concerning the role of the bed nucleus of the stria terminalis (BNST) in fear and anxiety-elicited behavior. Most of the studies investigating this role, however, employed irreversible lesions of this nucleus. The objective of the present study was to investigate the effects of an acute and reversible inactivation of the BNST in rats submitted to the Vogel conflict test (VCT) and contextual fear conditioning, two widely employed animal models that are responsive to prototypal anxiolytic drugs. Male Wistar rats were submitted to stereotaxic surgery to bilaterally implant cannulae into the BNST. Ten minutes before the test they received bilateral microinjections of cobalt chloride (COCl(2)) (1 mM/100 nL), a nonselective synapse blocker. COCl(2) produced anxiolytic-like effects in tests, increasing the number of punished licks in the VCT and decreasing freezing behavior and the increase in mean arterial blood pressure and heart rate of animals re-exposed to the context where they had received electrical foot shocks 24 h before. The results indicate that the BNST is engaged in behavioral responses elicited by punished stimuli and aversively conditioned contexts, reinforcing its proposed role in anxiety. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Activation of CB1 cannabinoid receptors in the dorsolateral periaqueductal gray reduces the expression of contextual fear conditioning in rats

Rationale Conditioned fear to context causes freezing and cardiovascular changes in rodents and has been used to measure anxiety. It also activates the dorsolateral column of the periaqueductal gray (dlPAG). Microinjections of cannabinoid agonists into the dlPAG produced anxiolytic-like effects in the elevated plus maze, but the effects of these treatments on fear conditioning remains unknown. Objective The objective of this study was to verify if intra-dlPAG injection of the CB1 cannabinoid receptor agonist anandamide (AEA) or the anandamide transport inhibitor AM404 would attenuate behavioral (freezing) and cardiovascular (increase of arterial pressure and heart rate) responses of rats submitted to a contextual fear-conditioning paradigm. Materials and methods Male Wistar rats with cannulae aimed at the dlPAG were re-exposed to a chamber where they had received footshocks 48 h before. Fifteen minutes before the test, the animals received a first intra-dlPAG injection of vehicle or AM251, a CB1 receptor antagonist (100 pmol/200 nl), followed 5 min later by vehicle, AEA (5 pmol/200 nl) or AM404 (50 pmol/200 nl). Freezing and cardiovascular responses were recorded for 10 min. Results Freezing and cardiovascular responses were reduced by administration of either AEA or AM404 into the dlPAG before re-exposition to the aversively conditioned context. These effects were abolished when the animals were locally pretreated with AM251. The latter drug, even at a higher dose (300 pmol), was ineffective when administered alone into the dlPAG. Conclusion The results suggest that facilitation of endocannabinoid-mediated neurotransmission in the dlPAG, through activation of local CB1 receptors, attenuates the expression of contextual fear responses.

Effects of reversible inactivation of the dorsal hippocampus on the behavioral and cardiovascular responses to an aversive conditioned context

In rats, conditioned fear to context causes freezing immobility and cardiovascular changes. The dorsal hippocampus (DH) has a critical role in several memory processes, including conditioning fear to contextual information. To explore a possible involvement of the DH in contextual fear conditioning-evoked cardiovascular (mean arterial pressure and heart rate increases) and behavioral (freezing) responses, DH synaptic transmission was temporarily inhibited by bilateral microinjections of 500 nl of the nonselective synapse blocker, cobalt chloride (COCl2, 1 mmol/l), at different periods of the experimental procedure. During re-exposure to the foot shock chamber in which conditioning had taken place, bilateral DH inhibition 10 min before the conditioning session had no effect on either behavioral or cardiovascular responses. Bilateral DH inhibition immediately after the conditioning session (110 min) decreased both behavioral and cardiovascular responses during the context test. Finally, 48 h after the conditioning session, bilateral DH inhibition 10 min before re-exposure to the foot shock chamber significantly reduced cardiovascular responses but not freezing responses. These results suggest that contextual fear conditioning acquisition does not depend on the DH. This structure, however, is crucial for the consolidation of contextual fear. Moreover, although the DH appears to be less important for the behavioral (freezing) changes induced by re-exposure to the aversive conditioned context, it may play an important role on the cardiovascular responses generated by this model.

Cannabinoid CB(1) receptors in the medial prefrontal cortex modulate the expression of contextual fear conditioning

The ventral portion of the medial prefrontal cortex (vMPFC) has been related to the expression of contextual fear conditioning. This study investigated the possible involvement of CB(1) receptors in this aversive response. Male Wistar rats were submitted to a contextual aversive conditioning session and 48 h later re-exposed to the aversive context in which freezing and cardiovascular responses (increase of arterial pressure and heart rate) were recorded. The expression of CB(1) receptor-mRNA in the vMPFC was also measured using real time-PCR. In the first experiment intra-vMPFC administration of the CB(1) receptor agonist anandamide (AEA, 5 pmol/200 nl) or the AEA transport inhibitor AM404 (50 pmol/200 nl) prior to re-exposure to the aversive context attenuated the fear-conditioned responses. These effects were prevented by local pretreatment with the CB(1) receptor antagonist AM251 (100 pmol/200 nl). Using the same conditioning protocol in another animal group, we observed that CB(1) receptor mRNA expression increased in the vMPFC 48 h after the conditioning session. Although AM251 did not cause any effect by itself in the first experiment, this drug facilitated freezing and cardiovascular responses when the conditioning session employed a lesser aversive condition. These results indicated that facilitation of cannabinoid-mediated neurotransmission in the vMPFC by local CB(1) receptor activation attenuates the expression of contextual fear responses. Together they suggest that local endocannabinoid-mediated neurotransmission in the vMPFC can modulate these responses.

Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats

Rationale: Systemic administration of cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa, is able to attenuate cardiovascular and behavioral (freezing) changes induced by re-exposure to a context that had been previously paired with footshocks. The brain sites mediating this effect, however, remain unknown. The medial prefrontal cortex (mPFC) has been related to contextual fear conditioning. Objectives: (1) To verify, using c-Fos immunocytochemistry, if the mPFC is involved in the attenuation of contextual fear induced by systemic administration of CBD; (2) to investigate if direct microinjections of CBD into mPFC regions would also attenuate contextual fear. Results: Confirming previous results systemic administration of CBD (10 mg/kg) decreased contextual fear and associated c-Fos expression in the prefrontal cortex (prelimbic and infralimbic regions). The drug also attenuated c-Fos expression in the bed nucleus of the stria terminalis (BNST). Direct CBD (30 nmol) microinjection into the PL prefrontal cortex reduced freezing induced by re-exposure to the aversively conditioned context. In the infralimbic (IL) prefrontal cortex, however, CBD (30 nmol) produced an opposite result, increasing the expression of contextual fear conditioning. This result was confirmed by an additional experiment where the conditioning session was performed under a less aversive protocol. Conclusion: These results suggest that the PL prefrontal cortex may be involved in the attenuation of contextual fear induced by systemic injection of CBD. They also support the proposition that the IL and PL play opposite roles in fear conditioning. A possible involvement of the BNST in CBD effects needs to be further investigated. (C) 2009 Elsevier B.V. All rights reserved.

Mild cognitive deficits associated to neocortical microgyria in mice with genetic deletion of cellular prion protein

The cellular prion protein (PrP(c)) has been implicated with the modulation of neuronal apoptosis, adhesion, neurite outgrowth and maintenance which are processes involved in the neocortical development. Malformations of cortical development (MCD) are frequently associated with neurological conditions including mental retardation, autism, and epilepsy. Here we investigated the behavioral performance of female adult PrP(c)-null mice (Prnp(%)) and their wild-type controls (Prnp(+/+)) presenting unilateral polymicrogyria, a MCD experimentally induced by neonatal freeze-lesion in the right hemisphere. injured mice from both genotypes presented similar locomotor activity but Prnp(%) mice showed a tendency to increase anxiety-related responses when compared to Prnp(+/+) animals. Additionally, injured Prnp(%) mice have a poorer performance in the social recognition task than sham-operated and Prnp(%) injured ones. Moreover the step-down inhibitory avoidance task was not affected by the procedure or the genotype of the animals. These data suggest that the genetic deletion of PrP(c) confers increased susceptibility to short-term social memory deficits induced by neonatal freezing model of polymicrogyria in mice. (C) 2008 Published by Elsevier B.V.

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin-like Growth Factor I, Insulin and Membrane Integrity of Bull Spermatozoa

Contents This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 mM and Glu 5 mM. In study II, semen was collected twice and divided into three aliquots; control, AA 50 mu M and AA 250 mu M. Initial sperm motility was significantly higher in sperm diluted with AA 50 mu M; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 mM.

Phase transitions of frozen camu-camu (Myrciaria dubia (HBK) McVaugh) pulp: Effect of cryostabilizer addition

Camu-camu is a tropical fruit with very high vitamin C content and commercialized as frozen pulp. Enthalpies of freezing, temperatures of the onset of ice melting, and glass transition temperatures of the maximally freeze-concentrated phase (T`(g)) of camu-camu pulp and of samples containing maltodextrin (DE20) and sucrose were measured by differential scanning calorimetry. Maltodextrin exhibited the largest freeze stabilization potential, increasing T`(g) from -58.2 degrees C (natural pulp) to -39.6 degrees C when 30% (w/w) maltodextrin DE 20 was added. Sucrose showed negligible effect on T`(g) but enhanced considerably the freezing point depression and less amount of ice was formed.

Contextual, but not auditory, fear conditioning is disrupted by neurotoxic selective lesion of the basal nucleus of amygdala in rats

The basolateral amygdala complex (BLA) is involved in acquisition of contextual and auditory fear conditioning. However, the BLA is not a single structure but comprises a group of nuclei, including the lateral (LA), basal (BA) and accessory basal (AB) nuclei. While it is consensual that the LA is critical for auditory fear conditioning, there is controversy on the participation of the BA in fear conditioning. Hodological and neurophysiological findings suggest that each of these nuclei processes distinct information in parallel; the BA would deal with polymodal or contextual representations, and the LA would process unimodal or elemental representations. Thus, it seems plausible to hypothesize that the BA is required for contextual, but not auditory, fear conditioning. This hypothesis was evaluated in Wistar rats submitted to multiple-site ibotenate-induced damage restricted to the BA and then exposed to a concurrent contextual and auditory fear conditioning training followed by separated contextual and auditory conditioning testing. Differing from electrolytic lesion and lidocaine inactivation, this surgical approach does not disturb fibers of passage originating in other brain areas, restricting damage to the aimed nucleus. Relative to the sham-operated controls, rats with selective damage to the BA exhibited disruption of performance in the contextual, but not the auditory, component of the task. Thus, while the BA seems required for contextual fear conditioning, it is not critical for both an auditory-US association, nor for the expression of the freezing response. (C) 2009 Elsevier Inc. All rights reserved.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.