RESUMO
Small bowel strictures remain a debilitating consequence of Crohn's disease and contribute to poor outcomes for patients. Recently, TGFß has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFß isoforms in ileal strictures of patients with Crohn's disease using in situ hybridization to understand TGFß's role in stricture formation. The mucosa of strictures was characterized by higher TGFß1 while the stricture submucosa showed higher TGFß3 compared to normal ileum from patients without Crohn's disease (p = 0.02 and p = 0.044, respectively). We correlated these findings with single-cell transcriptomics which demonstrated that TGFß3 transcripts overall are very rare, which may partially explain why its role in intestinal fibrosis has remained unclear to date. There were no significant differences in fibroblast or B cell TGFß1 and/or TGFß3 expression in inflamed vs. noninflamed ileum. We discuss the implications of these findings for therapeutic development strategies to treat patients with fibrostenotic Crohn's disease.
Assuntos
Doença de Crohn , Fibrose , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta3 , Humanos , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Doença de Crohn/complicações , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Constrição Patológica/metabolismo , Fibrose/metabolismo , Masculino , Feminino , Adulto , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Pessoa de Meia-Idade , Íleo/metabolismo , Íleo/patologiaRESUMO
The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-κB (NF-kB) and inhibitor κB (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.
Assuntos
Encéfalo/citologia , Núcleo Celular/metabolismo , Colostro , Neurônios/metabolismo , Ocitocina/metabolismo , Estresse Fisiológico , Animais , Animais Recém-Nascidos , Estresse do Retículo Endoplasmático , Feminino , Inflamação/metabolismo , Gravidez , Ratos , Transdução de SinaisRESUMO
Little is known about the role of oxytocin (OT) in colostrum during early gut colonization. We previously showed that transient OT receptor (OTR) expression on newborn rat enterocytes coincides with the milk-suckling period, and that OT activates endoplasmic reticulum stress sensors in cultured enterocytes. Here, we explored whether colostrum-OT attenuates stress in newborn villi primed and unprimed by colostrum by measuring levels of stress markers including BiP (an ER chaperone), eIF2a (translation initiation factor), and pPKR (eIF2a kinase). We also measured two inflammation-signaling proteins NF-κB and its inhibitor IκB. To test the impact of colostrum on autophagy, we measured a marker of autophagy initiation, LC3A. Colostrum increased inactive p-eIF2a, p-PKR and IκB and reduced p-IκB, BiP and LC3A. LPS increased and OT decreased p-IkB. BiP (GRP78) was higher in unprimed than primed villi. Together, these data suggest that colostrum OT attenuates the impact of inflammation on postnatal gut villi and that OT enhances autophagy to protect against amino acid insufficiency-induced stress during the interval between birth and the first feeding.
Assuntos
Colostro/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Mediadores da Inflamação/imunologia , Mucosa Intestinal/imunologia , Ocitocina/administração & dosagem , Animais , Animais Recém-Nascidos , Autofagia/efeitos dos fármacos , Autofagia/imunologia , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico/imunologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , Microvilosidades/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The neuropeptide neuromodulator and hormone oxytocin (OT) activates signaling pathways involved in mRNA translation in response to endoplasmic reticulum stress and reduces inflammation associated with experimental colitis in rats. The anti-inflammatory effects of OT may serve a vital role in the development, survival and function of newborn-type enterocytes during microbial gut colonization, which coincides with the milk suckling period when OT receptor expression peaks in the gut. Furthermore, mice deficient in the OT receptor have abnormal gut structure and function, underscoring OT's developmental importance. METHODS: We tested the effect of OT upon lipopolysaccharide (LPS)-induced markers of the inflammatory response in Caco2BB gut cells in vitro using automated immunocapillary electrophoresis. RESULTS: We demonstrate that OT suppresses NF-κB signaling and presumably inflammatory transcriptional programs, which are unleashed by LPS through the modulation of IκB. We show that OT counteracts LPS-elicited silencing of the unfolded protein response, a pathway limiting endoplasmic reticulum stress by suppressing protein translation. OT selectively activates dsRNA-activated kinase (PKR), X-box binding protein 1 (XBP1), immunoglobulin binding protein (BiP), A20 (TNFα-induced protein 3) and inositol requiring enzyme 1a (IRE1a). OT inactivates eukaryotic translation initiation factor 2a (eIF2a) without significant activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). CONCLUSIONS: Mild, preemptive stimulation of endoplasmic reticulum stress sensors by OT may precondition newborn enterocytes to resist apoptosis associated with inflammation and may support their differentiation and development by modulating cellular metabolism. GENERAL SIGNIFICANCE: OT may protect enterocytes and other cell types, such as neurons, from stress-related complications during postnatal development.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Proteínas de Choque Térmico/análise , Lipopolissacarídeos/antagonistas & inibidores , Ocitocina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células CACO-2 , Chaperona BiP do Retículo Endoplasmático , Humanos , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Resposta a Proteínas não DobradasRESUMO
BACKGROUND: Preterm infants are at high risk for adverse neurodevelopmental and behavioral outcomes. Family Nurture Intervention (FNI) in the Neonatal Intensive Care Unit (NICU) is designed to counteract adverse effects of separation of mothers and their preterm infants. Here, we evaluate effects of FNI on neurobehavioral outcomes. METHODS: Data were collected at 18 months corrected age from preterm infants. Infants were assigned at birth to FNI or standard care (SC). Bayley Scales of Infant Development III (Bayley-III) were assessed for 76 infants (SC, n = 31; FNI, n = 45); the Child Behavior Checklist (CBCL) for 57 infants (SC, n = 31; FNI, n = 26); and the Modified Checklist for Autism in Toddlers (M-CHAT) was obtained for 59 infants (SC, n = 33; FNI, n = 26). RESULTS: Family Nurture Intervention significantly improved Bayley-III cognitive (p = .039) and language (p = .008) scores for infants whose scores were greater than 85. FNI infants had fewer attention problems on the CBCL (p < .02). FNI improved total M-CHAT scores (p < .02). Seventy-six percent of SC infants failed at least one of the M-CHAT items, compared to 27% of FNI infants (p < .001). In addition, 36% of SC infants versus 0% of FNI infants failed at least one social-relatedness M-CHAT item (p < .001). CONCLUSIONS: Family Nurture Intervention is the first NICU intervention to show significant improvements in preterm infants across multiple domains of neurodevelopment, social-relatedness, and attention problems. These gains suggest that an intervention that facilitates emotional interactions between mothers and infants in the NICU may be key to altering developmental trajectories of preterm infants.
Assuntos
Desenvolvimento Infantil/fisiologia , Doenças do Prematuro/prevenção & controle , Recém-Nascido Prematuro/psicologia , Unidades de Terapia Intensiva Neonatal , Relações Mãe-Filho/psicologia , Psicoterapia/métodos , Adulto , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Resultado do TratamentoRESUMO
OBJECTIVE: This study assessed the impact of Family Nurture Intervention (FNI) on the quality of maternal caregiving behavior (MCB) while in the neonatal intensive care unit (NICU). FNI is a randomized controlled trial conducted in a high-acuity NICU to facilitate an emotional connection between mothers and their premature infants. FNI begins shortly after birth, continues until discharge, and involves mother/infant calming sessions that include scent cloth exchange, vocal soothing and emotion expression, eye contact, skin-to-skin and clothed holding, and family-based support sessions. METHODS: Maternal caregiving behavior was coded during a single holding and feeding session (â¼30 min) in the NICU before discharge at approximately 36 weeks gestational age (GA). Sixty-five mothers and their premature infants (34 male, 31 female; 26-34 wk GA) were included in these analyses (FNI, n = 35; standard care [SC], n = 30). RESULTS: Relative to mothers in the SC condition, those in the FNI group showed significantly higher quality MCB, which remained significant when controlling for birth order, twin status, maternal depression, and maternal anxiety. CONCLUSION: This is the first study to demonstrate that in-unit MCB can be enhanced by a hospital-based intervention. FNI provides a new rationale for integrating nurture-based interventions into standard NICU care.
Assuntos
Unidades de Terapia Intensiva Neonatal , Comportamento Materno , Relações Mãe-Filho , Mães/educação , Nascimento Prematuro/psicologia , Adulto , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Comportamento Materno/psicologia , Relações Mãe-Filho/psicologia , Mães/psicologiaRESUMO
We have shown that oxytocin receptor (OTR) expression in neonatal rat enterocytes is robust from birth to weaning, but OTR function during this period is unknown. We previously reported that oxytocin (OT) stimulation of Caco2BB cells (enterocytes in vitro) inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling. The unfolded protein response (UPR) is known to protectively reduce translation during endoplasmic reticulum (ER) stress. Because the mTORC1 pathway is linked to cellular stress, we investigated markers of UPR in OT-stimulated Caco2BB cells. We report that OT modulates several factors involved in sensing and translation of ER stress. High OT (62.5 nM) reduced translation initiation factor 4E-BP1 phosphorylation (Ser65), which is known to inhibit cap-dependent translation via its rate-limiting eukaryotic translation initiation factor 4E (eIF4E). Importantly, high OT increased phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) phospho-Ser51, which inhibits eIF2a. High OT also increased protein kinase RNA-like endoplasmic reticulum kinase phosphorylation, a sensor of ER stress and a kinase of eIF2a. Both high and low OT activated inositol requiring enzyme1 (IRE1), which generates the transcription factor X-box binding protein 1 (XBP1) and induces the UPR. We also show that OT modulates XBP1 splicing and induces tribbles 3 (TRIB3; a negative regulator of Akt and protein involved in autophagy) and immunoglobulin binding protein (BiP; ER-chaperone). Taken together, these results indicate that OT modulates sensors of ER stress and autophagy. These findings support our hypothesis that transiently elevated OTR expression in neonatal gut may serve a protective function during a critical postnatal developmental period.
Assuntos
Enterócitos/metabolismo , Enterócitos/patologia , Ocitocina/metabolismo , Resposta a Proteínas não Dobradas , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: While survival rates for preterm infants have increased, the risk for adverse long-term neurodevelopmental and behavioral outcomes remains very high. In response to the need for novel, evidence-based interventions that prevent such outcomes, we have assessed Family Nurture Intervention (FNI), a novel dual mother-infant intervention implemented while the infant is in the Neonatal Intensive Care Unit (NICU). Here, we report the first trial results, including the primary outcome measure, length of stay in the NICU and, the feasibility and safety of its implementation in a high acuity level IV NICU. METHODS: The FNI trial is a single center, parallel-group, randomized controlled trial at Morgan Stanley Children's Hospital for mothers and their singleton or twin infants of 26-34 weeks gestation. Families were randomized to standard care (SC) or (FNI). FNI was implemented by nurture specialists trained to facilitate affective communication between mother and infant during specified calming interactions. These interactions included scent cloth exchange, sustained touch, vocal soothing and eye contact, wrapped or skin-to-skin holding, plus family-based support interactions. RESULTS: A total of 826 infants born between 26 and 34 weeks during the 3.5 year study period were admitted to the NICU. After infant and mother screening plus exclusion due to circumstances that prevented the family from participating, 373 infants were eligible for the study. Of these, we were unable to schedule a consent meeting with 56, and consent was withheld by 165. Consent was obtained for 150 infants from 115 families. The infants were block randomized to groups of N = 78, FNI and N = 72, SC. Sixteen (9.6%) of the randomized infants did not complete the study to home discharge, 7% of those randomized to SC and 12% of FNI infants. Mothers in the intervention group engaged in 3 to 4 facilitated one- to two-hour sessions/week. Intent to treat analyses revealed no significant difference between groups in medical complications. The mean length of stay was not significantly affected by the intervention. CONCLUSION: There was no significant effect demonstrated with this intervention amount on the primary short-term outcome, length of stay. FNI can be safely and feasibly implemented within a level IV NICU. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01439269.
Assuntos
Recém-Nascido Prematuro/crescimento & desenvolvimento , Unidades de Terapia Intensiva Neonatal/organização & administração , Terapia Intensiva Neonatal/métodos , Tempo de Internação/estatística & dados numéricos , Comportamento Materno/psicologia , Relações Mãe-Filho/psicologia , Mães/educação , Relações Familiares , Estudos de Viabilidade , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro/psicologia , Análise de Intenção de Tratamento , Masculino , Mães/psicologia , Segurança do PacienteRESUMO
Our recent findings of a weaning-related pattern of oxytocin (OT) and OT receptor (OTR) expression in the rat enteric nervous system and in villus-crypt enterocytes, together with the known high level and stability of OT in breast milk support that OT may play a role in gut function and development. We previously described a biphasic dose-response of the PI3K/Akt pathway in gut cells treated with OT. Activation peaked at 62.5 nM OT (30 min) and coincided with OTR internalization. Here we use automated Western blotting to further explore OT-elicited changes in Akt and pAkt(T308), as well as in downstream substrates p70 S6 kinase-1 (S6K1) and eIF-4E binding protein 1 (4E-BP1). Relative to fresh growth medium (FGM) alone, our results showed OT in FGM reduced the abundance and phosphorylation of S6K1 and the phosphorylation of 4E-BP1, both substrates of mammalian target of rapamycin complex 1 (mTORC1). Phosphorylation of mTORC1 regulator, Raptor(S792), was increased by high and low OT concentrations, with predicted inhibitory effects on mTORC1. OT thus downregulates anabolic effects induced by FGM activity catalyzed by mTORC1. OT is a regulator of the PI3K/Akt/mTORC1 pathway in Caco2BB cells and may modulate translation in gut cells.
Assuntos
Trato Gastrointestinal/enzimologia , Complexos Multiproteicos/metabolismo , Ocitocina/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células CACO-2 , Fator de Iniciação 4E em Eucariotos/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Ocitocina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteína Regulatória Associada a mTOR , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In this exploratory, hypothesis-generating literature review, we evaluated potentially differential effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and non-HDL-C in published studies of ω-3 fatty acid supplementation or prescription ω-3 fatty acid ethyl esters. Placebo-adjusted changes in mean lipid parameters were compared in randomized, controlled trials in subjects treated for ≥ 4 weeks with DHA or EPA. Of 22 studies identified, 6 compared DHA with EPA directly, 12 studied DHA alone (including 14 DHA-treated groups), and 4 examined EPA alone. In studies directly comparing EPA with DHA, a net increase in LDL-C of 3.3% was observed with DHA (DHA: +2.6%; EPA: -0.7%). In such head-to-head comparative studies, DHA treatment was associated with a net decrease in TG by 6.8% (DHA: -22.4%; EPA: -15.6%); a net increase in non-HDL-C by 1.7% (DHA: -1.2%; EPA -2.9%); and a net increase in HDL-C by 5.9% (DHA: +7.3%; EPA: +1.4%). Increases in LDL-C were also observed in 71% of DHA-alone groups [with demonstrated statistical significance (P < .05) in 67% (8 of 12) DHA-alone studies] but not in any EPA-alone studies. Changes in LDL-C significantly correlated with baseline TG for DHA-treated groups. The range of HDL-C increases documented in DHA-alone vs EPA-alone studies further supports the fact that HDL-C is increased more substantially by DHA than EPA. In total, these findings suggest that DHA-containing supplements or therapies were associated with more significant increases in LDL-C and HDL-C than were EPA-containing supplements or therapies. Future prospective, randomized trials are warranted to confirm these preliminary findings, determine the potential effects of these fatty acids on other clinical outcomes, and evaluate the generalizability of the data to larger and more heterogeneous patient populations.
Assuntos
LDL-Colesterol/sangue , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Hiperlipidemias/sangue , Hipolipemiantes/farmacologia , Colesterol/sangue , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácido Eicosapentaenoico/uso terapêutico , Humanos , Hiperlipidemias/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Triglicerídeos/sangueRESUMO
Expression of cyclins D1 (cD1) and D2 (cD2) in ventricular zone and subventricular zone (SVZ), respectively, suggests that a switch to cD2 could be a requisite step in the generation of cortical intermediate progenitor cells (IPCs). However, direct evidence is lacking. Here, cD1 or cD2 was seen to colabel subsets of Pax6-expressing radial glial cells (RGCs), whereas only cD2 colabeled with Tbr2. Loss of IPCs in cD2(-/-) embryonic cortex and analysis of expression patterns in mutant embryos lacking cD2 or Tbr2 indicate that cD2 is used as progenitors transition from RGCs to IPCs and is important for the expansion of the IPC pool. This was further supported by the laminar thinning, microcephaly, and selective reduction in the cortical SVZ population in the cD2(-/-)cortex. Cell cycle dynamics between embryonic day 14-16 in knock-out lines showed preserved parameters in cD1 mutants that induced cD2 expression, but absence of cD2 was not compensated by cD1. Loss of cD2 was associated with reduced proliferation and enhanced cell cycle exit in embryonic cortical progenitors, indicating a crucial role of cD2 for the support of cortical IPC divisions. In addition, knock-out of cD2, but not cD1, affected both G(1)-phase and also S-phase duration, implicating the importance of these phases for division cycles that expand the progenitor pool. That cD2 was the predominant D-cyclin expressed in the human SVZ at 19-20 weeks gestation indicated the evolutionary importance of cD2 in larger mammals for whom expansive intermediate progenitor divisions are thought to enable generation of larger, convoluted, cerebral cortices.
Assuntos
Proliferação de Células , Córtex Cerebral/embriologia , Córtex Cerebral/fisiologia , Ciclinas/metabolismo , Células-Tronco/fisiologia , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Ciclo Celular/fisiologia , Ciclina D1/metabolismo , Ciclina D2 , Ciclinas/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Neuroglia/fisiologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de TempoRESUMO
G1-phase cyclin D1 (cD1) expression has been documented in post-mitotic neurons undergoing apoptosis, leading others to propose that attempted cell cycle re-entry may induce cell death. Here, cD1 immunoreactivity was found in a subpopulation of healthy excitatory neurons throughout the brain. Most striking was the selective cD1 expression in hippocampal pyramidal neurons, an especially vulnerable cell group. Seizure threshold, cD1 induction and CA1 neuron death were examined following application of kainate (KA) or pentylenetetrazole (PTZ) in cD1 heterozygous (+/-) and wildtype mice to determine whether baseline cD1 correlates with pathology. cD1+/- mice displayed resistance to KA, but not PTZ-induced seizures and had reduced or equivalent cytotoxicity respectively, compared with wildtype. KA administration, but not PTZ, induced cD1 expression. These findings suggest that basal cD1 expression may render hippocampal circuits more susceptible to particular epileptogenic agents and excitotoxic cell death, though cD1 is not a direct precipitant in apoptosis.
Assuntos
Apoptose/fisiologia , Ciclina D1/metabolismo , Epilepsia/metabolismo , Hipocampo/metabolismo , Degeneração Neural/metabolismo , Células Piramidais/metabolismo , Adulto , Animais , Apoptose/efeitos dos fármacos , Convulsivantes/farmacologia , Ciclina D1/genética , Modelos Animais de Doenças , Epilepsia/genética , Epilepsia/fisiopatologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Humanos , Imuno-Histoquímica , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Neurotoxinas/toxicidade , Pentilenotetrazol/farmacologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.
Assuntos
Córtex Cerebral/metabolismo , Ciclinas/genética , Interneurônios/metabolismo , Ácido gama-Aminobutírico/deficiência , Animais , Ciclo Celular/genética , Ciclina D2 , Feminino , Genes bcl-1/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Especificidade de Órgãos , Parvalbuminas/metabolismo , Gravidez , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Regulation of neural proliferation is an essential component of brain formation and is driven by both intrinsic cell cycle and extrinsic growth and trophic molecules. Among the cell cycle proteins, understanding of the relative roles of the G1-phase active cyclins D2 and D1 (cD2 and cD1) has been hampered by lack of data regarding their expression patterns. In this study, cD2 immunoreactivity was examined in the neocortex, ganglionic eminences/striatum, and hippocampal formation from embryonic day 12.5 until postnatal day 60 to more precisely characterize the expression of this protein during forebrain development. The localization of cD1 was also immunohistologically mapped for comparison. Throughout forebrain development, both overlapping and nonoverlapping protein expression of these cyclins suggests the presence of shared and unique cell cycle requirements for neurogenesis that distinguishes progenitor pools.
Assuntos
Ciclinas/genética , Prosencéfalo/citologia , Prosencéfalo/crescimento & desenvolvimento , Células-Tronco/fisiologia , Animais , Corpo Estriado/citologia , Corpo Estriado/embriologia , Corpo Estriado/crescimento & desenvolvimento , Ciclina D , Ciclina D2 , Ciclinas/metabolismo , Giro Denteado/citologia , Giro Denteado/embriologia , Giro Denteado/crescimento & desenvolvimento , Feminino , Fase G1/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Gravidez , Prosencéfalo/embriologia , Células-Tronco/classificaçãoRESUMO
Estrogens have antiinflammatory actions and protect the brain from ischemic injury. Cerebral ischemia is accompanied by an inflammatory reaction that contributes to the tissue damage, an effect mediated in part by toxic amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS). Therefore, estrogens may protect the female brain by modulating postischemic iNOS expression. To test this hypothesis, we studied whether iNOS plays a role in the mechanisms of the reduced susceptibility to ischemic injury observed in female mice. The middle cerebral artery was occluded for 20 mins using an intraluminal filament in C57Bl/6 mice, and infarct volume was assessed 3 days later in cresyl violet-stained sections. Infarcts were 53% smaller in female mice than in males (P < 0.05), a reduction abolished by ovariectomy (OVX) and reinstated by estrogen replacement. In normal female mice, postischemic iNOS mRNA was lower than in males (P < 0.05). Ovariectomy increased iNOS mRNA after ischemia and estrogen replacement blocked this effect. Furthermore, the iNOS inhibitor aminoguanidine reduced infarct volume in male, but not in female, mice. Similarly, male iNOS-null mice had smaller infarcts than wild-type mice, but female iNOS nulls were not protected. Ovariectomy and OVX with estrogen replacement did not affect infarct volume in iNOS-null female mice. The findings suggest that the neuroprotection conferred by estrogens is, in part, related to attenuation of iNOS expression. Such attenuation could result from the potent antiinflammatory effects of estrogens that downregulate iNOS expression via transcriptional or posttranscriptional mechanisms.
Assuntos
Infarto Cerebral/enzimologia , Ataque Isquêmico Transitório/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Infarto Cerebral/etiologia , Infarto Cerebral/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Guanidinas/farmacologia , Ataque Isquêmico Transitório/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Ovariectomia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , Fatores SexuaisRESUMO
Mice lacking dopamine D2 and D3 receptors and wildtype littermates were tested in a two-choice perceptual discrimination test that is dependent upon optimal functioning of the prefrontal cortex. Both mutants showed no deficits in attending to the perceptual stimuli or in shifting attention between stimulus dimensions. However, the performance of both mutants differed from the wildtype in different test phases. D2 mutants exhibited significant impairment in the first compound discrimination, indicating deficits in the initial acquisition of the task-governing rules. In contrast, D3 mutants performed significantly better in a set-shifting phase that required reversal learning. The higher response accuracy of D3 mutants was also accompanied by significantly increased response latency. A stereological assessment of test-induced expression of the c-fos gene in neurons of the anterior cingulate and prelimbic/infralimbic cortices revealed highest activation in D3 mutants, intermediate activation in wildtype and lowest activation in D2 mutants, indicating that response accuracy in the cognitive test correlates with the magnitude of prefrontal cortical activation regardless of which test phases revealed different performances. The study illustrates that dopamine differentially modulates prefrontal cortical activity during tasks requiring attention depending upon the type of D2-like receptor that is activated.
Assuntos
Atenção/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Receptores de Dopamina D2/genética , Animais , Transtornos Cognitivos/genética , Transtornos Cognitivos/fisiopatologia , Giro do Cíngulo/citologia , Giro do Cíngulo/fisiologia , Imuno-Histoquímica , Sistema Límbico/citologia , Sistema Límbico/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Córtex Pré-Frontal/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de Dopamina D3 , Reversão de Aprendizagem/fisiologiaRESUMO
Mice deficient for dopamine D2 and D3 receptors exhibit blunted D(1)-receptor responses to agonist stimulation. This blunted D1-receptor activity is prominent in the medial prefrontal cortex (mPFC) and results in a significantly impaired performance of the mutants in a test for spatial working memory. A single dose of methamphetamine (METH; 5 mg/kg i.p.), however, elicits a long-lasting increase in agonist-stimulated D1 receptor activity in the mPFC. In D2 mutants, this increase reaches wild-type levels, and the working memory of METH-treated mutants is completely rescued. In D3 mutants, however, the METH-induced increase in D1-receptor activity remains below wild-type levels and does not result in improved working memory performance. D2 and D3 mutants also differ in their locomotor responses to METH. Repeated administration of this drug (5 mg/kg administered three times at 2-h intervals) leads to a transition from horizontal hyperlocomotion to excessive orofacial stereotypy (taffy pulling) only in wild type and D3 mutants. In both genotypes, this transition is accompanied by a change in the relative ratios of striatal neuronal activation in two neurochemically distinct compartments, with striosomal neuronal activation exceeding that of the striatal matrix during stereotypy. Both the stereotypic response to METH and the associated predominant activation of neurons located in striosomes require D2-receptor expression. These studies indicate a differential requirement for D1- and D2-like receptor activation in mediating the effects of METH on cognitive and motor function.
Assuntos
Cognição/efeitos dos fármacos , Metanfetamina/farmacologia , Atividade Motora/efeitos dos fármacos , Receptores de Dopamina D2/deficiência , Animais , Cognição/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Receptores de Dopamina D2/biossíntese , Receptores de Dopamina D2/genética , Receptores de Dopamina D3RESUMO
Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal and D1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increased kinase-mediated phosphorylation.
Assuntos
Calcineurina/metabolismo , Receptores de Dopamina D1/metabolismo , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Inibidores de Calcineurina , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Toxina Pertussis/farmacologia , Fosfosserina/análise , Fosfosserina/metabolismo , Proteína Fosfatase 1 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Receptores de Dopamina D2/deficiência , Receptores de Dopamina D2/genéticaRESUMO
Stereotypic motor behavior is a widespread phenomenon of many neurologic and psychiatric disorders. Studies on the mechanisms controlling motor stereotypies have focused on the role of dopamine in modulating the activity of basal ganglia neuronal circuits, and recent results demonstrated that stereotypic motor responses characteristic of psychomotor stimulant sensitization correlate with an enhanced activation of neurons located in striatal striosomes that substantially exceeds that of the surrounding matrix. The present study tested whether predominant striosomal activation is a general predictor for stereotypy. Wild-type and dopamine D(2) and D(3) receptor knockout mice were treated either three times with methamphetamine (METH; 3 x 5 mg/kg every 2 hours) or once with a full D(1) agonist. Depending on the genotype, both treatments elicit the same focused stereotypy (taffy pulling). Repeated METH-treatment elicits intense stereotypy in wild-type and D(3) mutants but not in D(2) single and D(2)/D(3) double mutants. The stereotypic response of wild-type and D(3) mutants correlates with a predominant activation of neurons located in striosomes. No striosomal predominance is detected in METH-treated D(2) single and D(2)/D(3) double mutants. In contrast, D(2) single and D(2)/D(3) double mutants exhibited the most severe stereotypic response to D(1)-agonist treatment. However, this treatment did not result in enhanced striosomal activation. Thus, whereas the expression of stereotypy in response to repeated METH treatment requires D(2) receptor expression, D(2) receptor expression diminishes stereotypic responses to an acute dose of a D(1) agonist. Enhanced striosomal activation, however, is a reliable indicator of D(1)- and D(2)-receptor coactivation but not a predictor for repetitive motor behavior in general.
Assuntos
Doenças dos Gânglios da Base/metabolismo , Neostriado/metabolismo , Vias Neurais/metabolismo , Neurônios/metabolismo , Transtorno de Movimento Estereotipado/metabolismo , Animais , Doenças dos Gânglios da Base/genética , Doenças dos Gânglios da Base/fisiopatologia , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Masculino , Metanfetamina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neostriado/efeitos dos fármacos , Neostriado/fisiopatologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiopatologia , Neurônios/efeitos dos fármacos , Neurópilo/efeitos dos fármacos , Neurópilo/metabolismo , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/deficiência , Receptores de Dopamina D2/genética , Receptores de Dopamina D3 , Transtorno de Movimento Estereotipado/genética , Transtorno de Movimento Estereotipado/fisiopatologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
We investigated whether selective stimulation of neurons of the sympathoinhibitory ventral periaqueductal gray (VPAG), or sympathoexcitatory dorsal periaqueductal gray (DPAG), differentially modulates CBF and EEG and exerts neuroprotection. Electrical stimulation of either regions of PAG comparably elevated AP and CBF, whereas chemical stimulation with the D,L-homocysteine produced either sympathoinhibition accompanied by decrease in CBF from ventral region or sympathoexcitation accompanied by increase in CBF from dorsal region in nonspinalized rats. The CBF effects evoked from DPAG and VPAG by chemical stimulation were preserved in spinalized rats supporting that the evoked CBF responses result directly from stimulation and are not secondary to AP changes. Stimulation of either region, whether chemical or electrical, synchronized the EEG. To explore whether PAG stimulation might protect the brain against ischemic injury, in other rats the VPAG or DPAG were stimulated for 1 h (50 Hz, 1 s on/1 s off, 75-100 microA) and the middle cerebral artery occluded 72 h later. Stimulation of the DPAG, but not VPAG, significantly reduced infarction volumes relative to sham-stimulated controls as determined 24 h after occlusion. Elevations of AP and CBF did not differ between groups. We conclude: (a). intrinsic neurons of D- and VPAG differentially regulate CBF; (b). neurons of DPAG are neuroprotective independently of changes in CBF and/or AP. The DPAG effect on infarct volume may be related to the central neuroprotective pathway evoked by stimulation of the cerebellar FN.