Immobilization Research Articles

Stress‐induced Changes in Tyrosine Hydroxylase Gene Expression in Rat Hypothalamic Paraventricular, Periventricular, and Dorsomedial Nuclei

Particular hypothalamic nuclei--the paraventricular (PVN), periventricular (PeV), and dorsomedial (DMN)--are important centers regulating neuroendocrine and autonomic systems during stress. The present study investigated gene expression of catecholamines (CA)-synthesizing enzymes and protein levels, especially of tyrosine hydroxylase (TH), in response to acute (120 min) and repeated (7 x 120 min) immobilization (IMO) stress. Immunohistochemical staining revealed many TH-positive perikarya in the PeV, less in the DMN, and a few in the PVN. Analysis of microdissected tissue samples obtained from frozen brain sections by quantitative competitive PCR method revealed TH mRNA in the PVN, periventricular subdivision of the PVN (PePVN), and DMN. TH mRNA and TH protein levels were increased in all the nuclei investigated, in both acute and repeated stress conditions. Interruption of ascending CA pathways from the brain stem areas abolished TH gene expression in the PVN and DMN in response to stress. The present data suggest that cell groups expressing TH gene are present in all three hypothalamic nuclei studied and are highly activated by stress. The regulation of the majority of these TH-positive cells during stress is under modulatory control by brain stem or spinal cord areas.

Catecholamines and Estrogen Are Involved in the Pathogenesis of Emotional Stress‐induced Acute Heart Attack

Emotional stress triggers takotsubo cardiomyopathy in postmenopausal women. Clinical analysis of autonomic nervous function has revealed a transient increase of sympathetic nervous activity and decrease of vagal nervous activity. Immobilization (IMO) stress of rats can reproduce the electrocardiographic and left ventriculographic changes that occur in takotsubo cardiomyopathy, both of which are prevented by combined blockade of alpha- and beta-adrenoceptors. Estrogen supplementation partially attenuated these cardiac changes. It also attenuated the IMO-induced increase of c-Fos immunoreactivity, or c-fos mRNA expression in the lateral septum, medial amygdaloid nucleus, paraventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, laterodorsal tegmental nucleus, and locus ceruleus; these regions contain central sympathetic neurons and neurons with immunoreactive estrogen receptors. It also downregulated c-fos mRNA expression in the adrenal gland and the heart, suggesting an increase of estrogen attenuated the stress-induced hypothalamo-sympathoadrenal outflow from the central nervous system to the target organs. Estrogen treatment also upregulated the levels of cardioprotective substances, such as atrial natriuretic peptide and heat shock protein 70, in the heart. These data suggest that reduction of estrogen levels following menopause might be involved in the primary cause of takotsubo cardiomyopathy both by indirect action on the nervous system and by direct action on the heart.

A single exposure to immobilization causes long-lasting pituitary-adrenal and behavioral sensitization to mild stressors

We have previously reported that a single exposure to immobilization (IMO) in rats causes a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor. Since there are reports showing that a single exposure to other stressors causes sensitization of the HPA response to heterotypic stressors and increases anxiety-like behavior, we studied in the present work the long-term effects of IMO on behavioral and HPA response to mild superimposed stressors. In Experiments 1 and 2, adult male Sprague–Dawley rats were subjected to 2 h of IMO and then exposed for 5 min to the elevated plus-maze (EPM) at 1, 3 or 7 days after IMO. Blood samples were taken at 15 min after initial exposure to the EPM. Increases in anxiety-like behavior and HPA responsiveness to the EPM were found at all times post-IMO. Changes in the resting levels of HPA hormones did not explain the enhanced HPA responsiveness to the EPM (Experiment 3). In Experiments 4 and 5, we studied the effects of a single exposure to a shorter session of IMO (1 h) on behavioral and HPA responses to a brief and mild session of foot-shocks done 10 days after IMO. Neither previous IMO nor exposure to shocks in control rats modified behavior in the EPM. However, a brief session of shocks in previously IMO-exposed rats dramatically increased anxiety in the EPM. HPA and freezing responses to shocks were similar in control and previous IMO groups. Therefore, a single exposure to IMO appears to induce long-lasting HPA and behavioral sensitization to mild superimposed stressors, although the two responses are likely to be at least partially independent. Long-term effects of IMO on the susceptibility to stress-induced endocrine and emotional disturbances may be relevant to the characterization of animal models of post-traumatic stress.

Adrenocorticotropic hormone elevates gene expression for catecholamine biosynthesis in rat superior cervical ganglia and locus coeruleus by an adrenal independent mechanism

Classically, upon hypothalamic stimulation, adrenocorticotropic hormone (ACTH) is released from the pituitary and acts on melanocortin 2 receptors (MC2R) in the adrenal cortex, stimulating glucocorticoid synthesis and release. Our earlier studies suggested that ACTH might have a direct effect on sympathetic ganglia. To analyze further the involvement of ACTH in regulation of gene expression of norepinephrine (NE) biosynthetic enzymes, we examined the effect of bilateral adrenalectomy (ADX) of Sprague–Dawley male rats. Fourteen days post-ADX, as expected, plasma ACTH was elevated, and levels of tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and MC2R mRNAs in superior cervical ganglia (SCG), and TH mRNA in locus coeruleus (LC) were increased compared with sham-operated animals. To determine effect of pulsatile elevation of ACTH, corticosterone pellets were implanted to ADX rats. Similar to immobilization (IMO) stress ACTH injections to these animals caused a rise in ACTH in plasma and triggered elevation of TH and DBH mRNAs in SCG and in LC with single and repeated daily injections, and MC2R mRNA in SCG with single injections. To study the effect of ACTH in isolated cells, primary cultures of rat SCG were transfected with TH and DBH promoter constructs and treated with ACTH. In agreement with the in vivo data, ACTH elevated their promoter activities similar to levels triggered by cyclic AMP analog. ACTH in the human SK-N-SH neuroblastoma cells increased TH and DBH promoter activity and endogenous DBH mRNA levels. The results show that ACTH can have a direct effect on transcription and gene expression of NE biosynthetic enzymes even without contribution of adrenal hormones.

Low-volume muscular endurance and strength training during 3-week forearm immobilization was effective in preventing functional deterioration

PurposeThe purpose of this study was to determine whether endurance and strength hand grip exercises during 3-week upper limb immobilization preserve muscle oxidative capacity, endurance performance and strength.MethodsTen healthy adult men underwent non-dominant forearm immobilization by plaster cast for 21 days. Five healthy adult subjects were designated as the immobilization (IMM) group and five were designated as the immobilization + training (IMM+TRN) group. Grip strength, forearm circumference, dynamic handgrip endurance and muscle oxygenation response were measured before and after the 21 day immobilization period. Using near-infrared spectroscopy (NIRS), muscle oxygen consumption recovery (VO2mus) was recorded after a submaximal exercise and the recovery time constant (TcVO2mus) was calculated. Reactive hyperemic oxygenation recovery was evaluated after 5 minutes ischemia. Two training programs were performed by the IMM+TRN group twice a week. One exercise involved a handgrip exercise at 30% maximum voluntary contraction (MVC) at a rate of 1 repetition per 1 second until exhaustion (about 60 seconds). The other involved a handgrip exercise at 70% MVC for 2 seconds with a 2 second rest interval, repeated 10 times (40 seconds).ResultsThere was a significant group-by-time interaction between the IMM and IMM+TRN groups in the TcVO2mus (p = 0.032, F = 6.711). A significant group-by-time interaction was observed between the IMM and IMM+TRN groups in the MVC (p = 0.001, F = 30.415) and in grip endurance (p = 0.014, F = 9.791). No significant group-by-time interaction was seen in forearm circumference and reactive hyperemic oxygenation response either in IMM or IMM+TRN group.ConclusionThe training programs during immobilization period used in this experiment were effective in preventing a decline in muscle oxidative function, endurance and strength.

Nitric Oxide and the Release of Lipoprotein Lipase from White Adipose Tissue

Background/Aim: Lipoprotein lipase (LPL) is the main enzyme responsible for the distribution of circulating triacylglycerides in tissues. Its regulation via release from active sites in the vascular endothelium is poorly understood. In a previous study we reported that in response to acute immobilization (IMMO), LPL activity rapidly increases in plasma and decreases in white adipose tissue (WAT) in rats. In other stress situations IMMO triggers a generalized increase in nitric oxide (NO) production. Methods/Results: Here we demonstrate that in rats: 1) in vivo acute IMMO rapidly increases NO concentrations in plasma 2) during acute IMMO the WAT probably produces NO via the endothelial isoform of nitric oxide synthase (eNOS) from vessels, and 3) epididymal WAT perfused in situ with an NO donor rapidly releases LPL from the endothelium. Conclusion: We propose the following chain of events: stress stimulus / rapid increase of NO production in WAT (by eNOS) / release of LPL from the endothelium in WAT vessels. This chain of events could be a new mechanism that promotes the rapid decrease of WAT LPL activity in response to a physiological stimulus.

Immobilization: Yay or nay?

Immobilization: Yay or nay?

Gen-X Immobilization

Gen-X Immobilization

THE CLASSIC: Wiring of the Vertebrae as a Means of Immobilization in Fracture and Potts' Disease

THE CLASSIC: Wiring of the Vertebrae as a Means of Immobilization in Fracture and Potts' Disease

Stress-induced alterations in catecholamine enzymes gene expression in the hypothalamic dorsomedial nucleus are modulated by caudal brain and not hypothalamic paraventricular nucleus neurons

The hypothalamic dorsomedial nucleus (DMN) represents an important coordinate center for regulation of autonomic and neuroendocrine systems, especially during stress response. The present study was focused on the gene expression of catecholamine-synthesizing enzymes and the protein levels of tyrosine hydroxylase in DMN, both in control and stressed rats. Moreover, pathways modulating the gene expression of tyrosine hydroxylase in DMN during immobilization (IMO) stress were also investigated. Gene expressions of all catecholamine-synthesizing enzymes were detected in DMN samples. While the levels of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA were increased in IMO rats, aromatic l-amino acid decarboxylase and dopamine-β-hydroxylase mRNA remained unchanged. Tyrosine hydroxylase protein levels were significantly elevated in the DMN only after repeated IMO stress. Postero-lateral deafferentations of the DMN, or transections of the ascending catecholaminergic pathways originating in the lower brainstem abolished the IMO-induced increase of tyrosine hydroxylase gene expression in the DMN. Nevertheless, postero-lateral deafferentations of the hypothalamic paraventricular nucleus (PVN), which separate the DMN from the PVN, had no effect on IMO-induced elevation of tyrosine hydroxylase mRNA in the DMN. The present data indicate that certain DMN neurons synthesize mRNA of catecholamine enzymes. The stress-induced increase of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA in DMN neurons indicates the involvement of these catecholaminergic neurons in stress response. The gene expression of tyrosine hydroxylase in DMN is modulated by lower brainstem and/or spinal cord, but not by PVN afferents.

Fungal laccase: copper induction, semi-purification, immobilization, phenolic effluent treatment and electrochemical measurement

The laccase activity induced by copper ions in Trametes versicolor by two inducers was studied and the aims were to show that it is possible to obtain a high laccase activity in almost 12 of the enzymes product using a basic culture medium but different copper concentration. Laccase was inducted by 2,5-xylidine and copper sulfate. Semi-purification of the crude laccase was carried out through precipitation and the column separation with NaCl gradient. In order to apply in an effluent treatment, laccase was immobilized on different vitroceramics supports, pyrolytic graphite and also on a carbon fiber electrode as biosensor. The maximum laccase activity was 40,774.0 U L-1 at the 12th day. The best support for immobilization was pyrolytic graphite (glutaraldehyde treated-94% efficiency). Total phenol removal in Kraft E1 effluent was 19% for immobilized pyrolytic graphite with the mediator present. The biosensor prepared with this material showed a good linear response to catechol. The optimization of laccase activity induction through the combination of 2,5-xylidine and cooper sulfate was obtained and led to its use in environmental remediation. Key words: Trametes versicolor, laccase induction, semi-purification, immobilization, effluent treatment.

Retroperitoneal white adipose tissue lipoprotein lipase activity is rapidly down-regulated in response to acute stress

Tissue-specific regulation of LPL has been widely studied in rats. Previous studies reported that in vivo administration of adrenaline and acute stress cause an increase in plasma LPL activity coinciding with a decrease in white adipose tissue (WAT) LPL activity. We studied the speed of LPL activity changes during 30 min of stress by immobilization (IMMO) in rats. A first experimental approach in permanently cannulated rats permitted sequential blood sampling in the same animal during IMMO and the obtaining of hemodynamic parameters. In a second experimental approach, animals were euthanized at different times after the start of IMMO to determine LPL activity in tissues. Stress was characterized by rises in blood pressure, heart rate, plasma corticosterone, and available circulating energy substrates. Five min after the start of IMMO, LPL activity fell in retroperitoneal WAT and increased in plasma. These data show the quickest LPL activity change ever described in response to a physiological situation. The speed and simultaneity of these changes suggest that the release from endothelium to the bloodstream may constitute a fast nonexplored mechanism of tissue LPL activity regulation, involved in the lipid energy-substrate redistribution between tissues needed to prepare the "fight-or-flight" response.

Stress‐induced changes in epinephrine expression in the adrenal medulla in vivo

Immobilization (IMMO) stress was used to examine how stress alters the stress hormone epinephrine (EPI) in the adrenal medulla in vivo. In rats subjected to IMMO for 30 or 120 min, adrenal corticosterone increased to the same extent. In contrast, EPI changed very little, suggesting that EPI synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological responses of the 'flight or fight' response. In part, stress activates EPI via the phenylethanolamine N-methyltransferase (PNMT) gene as single or repeated IMMO elevated PNMT mRNA. The rise in PNMT mRNA was preceded by induction of the PNMT gene activator, Egr-1, with increases in Egr-1 mRNA, protein, and protein-DNA binding complex apparent. IMMO also evoked changes in Sp1 mRNA, protein, and Sp1-DNA complex formation, although for chronic IMMO changes were not entirely coincident. In contrast, glucocorticoid receptor and AP-2 mRNA, protein, and protein-DNA complex were unaltered. Finally, IMMO stress elevated PNMT protein. However, with seven daily IMMOs for 120 min and delayed killing, protein stimulation did not attain the highly elevated levels expected based on mRNA changes. The latter may perhaps suggest initiation of adrenergic desensitization to prolonged and repeated IMMO stress and/or dissociation of transcriptional and post-transcriptional regulatory mechanisms.

Differential effects of stress and amphetamine administration on Fos‐like protein expression in corticotropin releasing factor‐neurons of the rat brain

Corticotropin releasing factor (CRF) appears to be critical for the control of important aspects of the behavioral and physiological response to stressors and drugs of abuse. However, the extent to which the different brain CRF neuronal populations are similarly activated after stress and drug administration is not known. We then studied, using double immunohistochemistry for CRF and Fos protein, stress and amphetamine-induced activation of CRF neurons in cortex, central amygdala (CeA), medial parvocellular dorsal, and submagnocellular parvocellular regions of the paraventricular nucleus of the hypothalamus (PVNmpd and PVNsm, respectively) and Barrington nucleus (Bar). Neither exposure to a novel environment (hole-board, HB) nor immobilization (IMO) increased Fos-like immunoreactivity (FLI) in the CeA, but they did to the same extent in cortical regions. In other regions only IMO increased FLI. HB and IMO both failed to activate CRF+ neurons in cortical areas, but after IMO, some neurons expressing FLI in the PVNsm and most of them in the PVNmpd and Bar were CRF+. Amphetamine administration increased FLI in cortical areas and CeA (with some CRF+ neurons expressing FLI), whereas the number of CRF+ neurons increased only in the PVNsm, in contrast to the effects of IMO. The present results indicate that stress and amphetamine elicited a distinct pattern of brain Fos-like protein expression and differentially activated some of the brain CRF neuronal populations, despite similar levels of overall FLI in the case of IMO and amphetamine.

Estimulação elétrica neuromuscular em cães submetidos à imobilização rígida temporária da articulação fêmoro-tíbio-patelar

Com o objetivo de avaliar o efeito da estimulação elétrica neuromuscular (EENM) de baixa freqüência sobre o músculo vasto lateral, foram utilizados 11 cães, agrupados aleatoriamente em três grupos, denominados de I ou controle, de II (EENM após imobilização) e de III (EENM durante e após imobilização). A articulação fêmoro-tíbio-patelar direita foi imobilizada por 30 dias pelo método de transfixação percutânea tipo II. Os cães do grupo III iniciaram as sessões de eletroterapia, três vezes por semana, durante (30 dias) e após a imobilização (60 dias); e os cães dos grupos II após a remoção da imobilização rígida temporária. Foram avaliadas a mensuração da circunferência da coxa, a goniometria do joelho, os graus de claudicação, as enzimas creatina-quinase (CK) e a aspartato-amino-transferase (AST) e a morfometria das fibras musculares longitudinais do vasto lateral. A análise clínica dos graus de claudicação foi realizada diariamente. A medida da circunferência de coxa, a goniometria e a biópsia do músculo vasto lateral foram realizadas nos tempos zero, 30, 60 e 90 dias após imobilização. As amostras de sangue para avaliação da CK e da AST foram coletadas antes, imediatamente depois, em 6, 24 e 48 horas após a EENM, nos dias zero, 1, 7, 30, 45, 60, 75 e 90. A EENM foi empregada no músculo quadríceps femoral numa freqüência de 50Hz, com duração de pulso de 300 milisegundos e relação "on time/off time" de 1:2. Não houve diferença significativa nos graus de claudicação, nos valores de circunferência da coxa, na goniometria e no comportamento das enzimas CK e AST entre os grupos. Foi observada maior hipertrofia das fibras musculares longitudinais nos cães do grupo II (P=0,0005), seguidos pelos cães do grupo III. Pode-se concluir que a EENM de baixa freqüência ocasiona hipertrofia do músculo vasto lateral em cães após a imobilização rígida temporária da articulação do joelho, recomendando-se o seu uso em animais com atrofia muscular.

アゾポリマーの光応答性を利用した微小球の配列と位置選択的固定

アゾポリマーの光応答性を利用した微小球の配列と位置選択的固定

Effects of prenatal stress on male offspring sexual maturity

Prenatal stimulations have been shown to have long-term effects on at reproductive activity. We evaluated the influence of the prenatal stress on the hypothalamic-pituitary-gonad (HPG) axis in male offsprings from mothers with high number of offsprings per litter (HNL) and low number of offsprings per litter (LNL) after hypothesizing that the number of offsprings per litter may modify the effect of the prenatal stress on the HPG of adult offsprings. Pregnant Wistar rats were used for this study. Immobilization (IMO) stress was used, 30 min, 3 times per week, from the 5th to 21st day of pregnancy. The weight of adrenal and gonads, and the corticosterone (COR), testosterone (TES) and luteinizing hormone (LH) plasmatic levels were analyzed in the male offspring at 30, 45 and 70 days of age. The offspring males coming from LNL showed a decrease in testicle weight and TES levels, without changes in the plasmatic LH levels. However, the offspring of HNL showed a decrease of LH levels. It is possible to conclude that in LNL prenatal stress would produce alterations to gonadal level, while in HNL the effect of stress would be evident at pituitary level.

Dynamics of immediate early gene and neuropeptide gene response to prolonged immobilization stress: evidence against a critical role of the termination of exposure to the stressor

Stress-induced expression of immediate early genes (IEGs) appears to be transient even if the exposure to the stressor persists. However, there are some exceptions which suggest that particular characteristics of stressors can affect the dynamics of IEG expression. We studied in selected telencephalic, diencephalic and brainstem regions the mRNA levels of two clearly distinct IEGs (c-fos and arc) during prolonged exposure to a severe stressor such as immobilization (IMO) and after releasing the rats from the situation. Although regional differences were observed with the two IEGs, overall, c-fos mRNA levels progressively declined over the course of 4 h of continuous exposure to IMO, whereas arc mRNA levels were maintained at high levels in the brain regions that express this gene under stress (telencephalon). Levels of CRF hnRNA in the hypothalamus paraventricular nucleus only slightly declined during prolonged exposure to IMO. Surprisingly, termination of exposure to IMO did not modify CRF gene expression in the paraventricular nucleus or the pattern of IEGs expression, with the exception of c-fos in the lateral septum. Thus, putative signals associated to the termination of exposure to IMO were unable to modify either IEG expression in most brain areas or CRF gene expression in the paraventricular nucleus.

Long Durations of Immobilization in the Rat Result in Enhanced Mechanical Properties of the Healing Supraspinatus Tendon Insertion Site

Rotator cuff tears frequently occur and can lead to pain and decreased shoulder function. Repair of the torn tendon back to bone is often successful in relieving pain, but failure of the repair commonly occurs. Post-operative activity level is an important treatment component that has received minimal attention for the shoulder, but may have the potential to enhance tendon to bone healing. The objective of this study was to investigate the effect of short and long durations of various activity levels on the healing supraspinatus tendon to bone insertion site. Rotator cuff tears were surgically created in Sprague-Dawley rats by detaching the supraspinatus tendon from its insertion on the humerus and these tears were immediately repaired back to the insertion site. The post-operative activity level was controlled through shoulder immobilization (IM), cage activity (CA), or moderate exercise (EX) for durations of 4 or 16 weeks. The healing tissue was evaluated utilizing biomechanical testing and a quantitative polarized light microscopy method. We found that activity level had no effect on the elastic properties (stiffness, modulus) of the insertion site at four weeks post injury and repair, and a decreased activity level had a positive effect on these properties at 16 weeks (IM>CA=EX). Furthermore, a decreased activity level had the greatest positive effect on these properties over time (IM>CA=EX). The angular deviation of the collagen, a measure of disorganization, was decreased with a decrease in activity level at 4 weeks (IM<CA=EX), but was similar between groups at 16 weeks (IM=CA=EX). It appears from this study that decreasing the activity level by immobilizing the shoulder improves tendon to bone healing, which progresses by first increasing the organization of the collagen and then increasing the mechanical properties. Future studies in this area will investigate the effect of passive motion and remobilization on both tendon to bone healing and shoulder function.

Interstate Immobilization

Interstate Immobilization