Immobilization Research Articles

Effect of acute and chronic immobilization stress on plasma levels of nicorandil administered orally to rats.

Effects of acute (15h) and chronic (15h x 7 days) immobilization (IM) stress on plasma levels of nicorandil [N-(2-hydroxyethyl) nicotinamide nitrate (ester)] administered orally were examined in rats. The maximum plasma level was reached 30 min after administration. Acute IM stress significantly reduced plasma nicorandil levels both in the absorption and elimination phases (15 min and 2-6h after administration, respectively). Chronic IM stress further intensified the reduction of nicorandil levels in the absorption phase, but attenuated the influence of acute stress in the elimination phase. No significant difference was observed one day after removal of chronic IM stress. These results suggest that chronic IM stress markedly inhibits the absorption of nicorandil, but the distribution, metabolism and excretion were influenced more by acute IM stress.

Immobilization stress may increase plasma interleukin-6 via central and peripheral catecholamines.

It has recently been reported that both physical and psychological stress elevate plasma interleukin (IL)-6 levels independently of endotoxemia, tissue damage, or inflammation. However, the mechanism of plasma IL-6 elevation in these models is poorly understood. In the present study, plasma IL-6 levels were measured using the IL-6-dependent murine hybridoma subclone B9 cell line, which is commonly used by other investigators. We first demonstrated that an immobilization (IM) stress, a typical physicopsychological stress, increased plasma IL-6 levels. Then the contribution of the hypothalamic-pituitary-adrenal (HPA) axis and the central and peripheral catecholaminergic systems in IM-induced plasma IL-6 elevation were examined because these mechanisms play important roles in host defense against stress. Blood samples were collected through an indwelling jugular venous catheter before, during, and after IM; the number of samples taken serially from each animal was 12-13. Blood cells were resuspended in a saline solution and injected into the animals through the same catheter after each blood collection in order to prevent loss of blood volume. After initiation of restraint, plasma IL-6 levels significantly increased at 60 min and peaked at 90 min in the animals immobilized for either 30 or 120 min. The peak levels of IM-induced plasma IL-6 in the animals immobilized for 120 min (1,905 +/- 414 U/ml) were significantly higher than those in the animals subjected to 30 min IM (837 +/- 95 U/ml; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of acute and chronic ACTH administration on pituitary-adrenal response to acute immobilization stress. Relationship to changes in corticosteroid-binding globulin.

The effect of single and chronic ACTH administration on serum levels of the corticosteroid-binding globulin (CBG) and pituitary-adrenal (PA) responsiveness to acute immobilization (IMO) stress was studied in adult Sprague-Dawley rats. Single ACTH administration significantly reduced CBG levels but did not alter PA response to acute IMO. Chronic ACTH administration caused a greater fall in CBG than single ACTH administration and blunted PA response to IMO. The effect of chronic ACTH administration on CGB levels recovered 2 days after the last administration, but the ACTH response to IMO was normal only by day 7 after the last ACTH injection. The present data indicate that ACTH administration to rats reduced CBG levels and impaired PA response to acute stress, but impaired PA responsiveness cannot be solely attributed to changes in CBG.

Direct evidence for chronic stress-induced facilitation of the adrenocorticotropin response to a novel acute stressor.

The ACTH response to CRF and the role of glucocorticoids on the pituitary-adrenal responsiveness to acute stressors after a period of chronic stress were assessed in Sprague-Dawley rats. After chronic immobilization (IMO) an enhanced ACTH response to CRF administration was observed. In another experiment, control and chronic IMO rats were adrenalectomized (ADX) or sham-adrenalectomized (SHAM) and 2 days later killed in resting conditions or after having been subjected to acute IMO or tail-shock for 30 min. Chronic IMO increased basal corticosterone but not adrenocorticotropin (ACTH) levels in SHAM rats. As expected, ADX increased ACTH levels in all conditions. In response to the novel acute stressor (shock), ACTH levels were drastically dependent on the presence of corticosterone: thus, whereas in SHAM rats chronic IMO reduced the ACTH response to shock, in ADX rats a clear enhancement of the ACTH response to shock was observed in chronic IMO rats. In order to demonstrate that, in our experimental conditions, chronic stress also induces down-regulation of glucocorticoid receptors in the hippocampus, an additional experiment was done: rats subjected chronically to IMO or administered 5 mg corticosterone (B) were adrenalectomized and killed 20 h later under basal conditions. Both chronic IMO and chronic B administration decreased glucocorticoid type II binding in the hippocampus. From these results, it is concluded that chronic IMO induces facilitation of the ACTH response to novel acute stressors which is uncovered after corticosterone removal.

Ventromedial hypothalamus mediates stress-induced hypocalcemia via the gastric vagus in rats

A hypothalamo-vagal mechanism of immobilization (IMB) stress-induced hypocalcemia was investigated in rats. Bilateral lesions in the Ventromedial nucleus of the hypothalamus (VMH), but not those of the lateral hypothalamic area (LHA) or the paraventricular nucleus (PVN), eliminated the calcium-lowering effect of IMB. None of these lesions, however, affected the basal levels of the blood calcium. An electrical stimulation of the VMH induced a significant decrease in the blood calcium level (0.07 mM fall) 60 min after stimulation. The hypocalcemic response was eliminated by a vagotomy of the gastric branches but not by that of the thyroid/parathyroid branches. These results suggest that the VMH mediates IMB-induced hypocalcemia through its influence on the gastric vagus.

The stomach is the etiologic organ for immobilization-induced hypocalcemia in rats.

Our previous study revealed that the gastric vagus nerve plays an etiologic role in immobilization (IMB) stress-induced hypocalcemia. The purpose of the present study is to identify exactly what parts of the stomach are involved in the development of IBM-induced hypocalcemia and to determine whether or not gastric acid secretion is involved. A total gastrectomy, but not a resection of the upper intestine, eliminated the hypocalcemic effect of IMB. In addition, either an antrectomy (removal of the source of gastrin) or a fundectomy (depriving the origin of gastric histamine and gastric acid) was sufficient for eliminating IMB-induced hypocalcemia, while a partial (50%) fundectomy failed to suppress it. An intraperitoneal injection of galanin (an inhibitor of gastrin release) or ranitidine (a blocker of histamine H2-receptor) also suppressed the calcium-lowering effect of IBM, whereas omeprazole (an inhibitor of the proton pump) had no effect. These findings suggest that the antrum and the fundus of the stomach play essential roles in IMB-induced hypocalcemia through the vagus-induced release of gastrin and histamine but not through the secretion of gastric acid per se.

Comparative morphometric changes in rat cortical bone following ovariectomy and/or immobilization

Gonadal hormone deficiency following ovariectomy and skeletal unloading by limb immobilization are useful models of osteopenia. The purpose of this study was to compare changes in cortical bone after ovariectomy (OVX) or immobilization (IMM) for 6 and 12 weeks. Comparisons were also made when rats were ovariectomized or immobilized for 6 weeks and then immobilized (OVX/IMM) and ovariectomized (IMM/OVX), respectively, for 6 more weeks. Tibias and femurs were collected and static and dynamic cortical bone indices were determined by morphometric methods. Femurs from animals OVX or IMM for 12 weeks were tested for bone stiffness by torsional testing. Six and 12 weeks after OVX, there were increases in the periosteal perimeter, cortical area, and periosteal bone formation indices, indicating that ovariectomy increased modeling-dependent bone gain on the periosteal envelope, relative to controls. Contrarily, 6 and 12 weeks after IMM, there were decreases, compared with controls, in periosteal perimeter, cortical bone area, and periosteal bone formation indices. This indicates that immobilization decreased modeling-dependent bone gain on the periosteal envelope. These differences in modeling between the animals that were OVX and IMM resulted in a smaller cortical width and minimum cortical width in the IMM compared with the OVX animals. There were significant decreases in cortical bone stiffness and minimum cortical width at the fracture site following mechanical testing in the animals IMM for 12 weeks. Both ovariectomy and immobilization increased endocortical resorption surface, endocortical perimeter and expansion of the marrow cavity. Because of suppressed periosteal bone formation with increased endocortical resorption, immobilization had a greater effect on bone loss and decreased bone stiffness than did ovariectomy. In the OVX/IMM or IMM/OVX groups, there were changes that reflected both conditions. Immobilization mitigated the increase in periosteal bone formation but tended to augment endocortical resorption following ovariectomy. These results show that ovariectomy and immobilization have envelope-specific effects on rat cortical bone.

Adrenalectomy augments in vivo release of norepinephrine in the paraventricular nucleus during immobilization stress.

Adrenalectomy (ADX) activates CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus and increases hypothalamic norepinephrine (NE) turnover in vitro. Immobilization (IMMO) markedly increases the release of NE into extracellular fluid in the PVN. The present study assessed whether ADX affects the release of NE in the PVN in vivo in conscious rats at baseline and during IMMO and whether cortisol (CORT) treatment attenuates the effects of ADX. Concentrations of NE, the NE metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol, and the dopamine metabolite dihydroxyphenylacetic acid were measured in microdialysate samples beginning 24 h after implantation of a microdialysis probe in the PVN. Seven to 10 days after ADX or ADX plus CORT (20 mg/kg.day via osmotic minipump), animals underwent IMMO for 2 h. Adrenalectomized rats had slightly higher baseline microdialysate NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, and dihydroxyphenylacetic acid levels and much larger IMMO-induced responses of these compounds than did sham-operated rats. CORT treatment abolished the ADX-induced augmentation of these responses. The results indicate that ADX, by removing endogenous glucocorticoids, augments IMMO-induced release and turnover of NE and amplifies the responses of catecholamine synthesis. Glucocorticoids, therefore, appear to exert feedback inhibition on stress-induced increments in the release of NE and catecholamine biosynthesis in the PVN.

Gastric vagus mediates immobilization-induced hypocalcemia in rats.

The involvement of the parasympathetic nervous system in the etiology of stress-induced hypocalcemia was investigated in the rat. Atropine methyl bromide (0.1 and 0.6 mg/kg ip) given 20 min before immobilization (IMB) was observed to suppress the induction of hypocalcemia in a dose-dependent manner. A vagotomy of the bilateral cervical trunks also abolished the IMB-induced hypocalcemia. A vagotomy on either the thyroid/parathyroid branches or the celiac branches had no effect on the IMB-induced hypocalcemia, but a vagotomy on the gastric branches completely abolished it. Pretreatment with either secretin (2 and 6 micrograms/kg ip), an inhibitor of gastrin release, or cimetidine (5 and 10 mg/kg ip), a histamine H2-receptor antagonist, diminished the IMB-induced hypocalcemia. The concentration of serum gastrin increased significantly during IMB. It is thus concluded that the decreased levels of plasma calcium caused by IMB are due to the activation of the vagus innervating the stomach. Gastrin and histamine are also involved as a consequence of the activation of the vagus.

Adrenalectomy augments in vivo release of norepinephrine in the paraventricular nucleus during immobilization stress

Adrenalectomy (ADX) activates CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus and increases hypothalamic norepinephrine (NE) turnover in vitro. Immobilization (IMMO) markedly increases the release of NE into extracellular fluid in the PVN. The present study assessed whether ADX affects the release of NE in the PVN in vivo in conscious rats at baseline and during IMMO and whether cortisol (CORT) treatment attenuates the effects of ADX. Concentrations of NE, the NE metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol, and the dopamine metabolite dihydroxyphenylacetic acid were measured in microdialysate samples beginning 24 h after implantation of a microdialysis probe in the PVN. Seven to 10 days after ADX or ADX plus CORT (20 mg/kg.day via osmotic minipump), animals underwent IMMO for 2 h. Adrenalectomized rats had slightly higher baseline microdialysate NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, and dihydroxyphenylacetic acid levels and much larger IMMO-induced responses of these compounds than did sham-operated rats. CORT treatment abolished the ADX-induced augmentation of these responses. The results indicate that ADX, by removing endogenous glucocorticoids, augments IMMO-induced release and turnover of NE and amplifies the responses of catecholamine synthesis. Glucocorticoids, therefore, appear to exert feedback inhibition on stress-induced increments in the release of NE and catecholamine biosynthesis in the PVN.

Chronic but not Acute Exposure to Stress is Associated with Hypothalamic Vasoactive Intestinal Polypeptide (VIP) Release into Median Eminence

The influence of stress on hypothalamic VIP release into the pituitary portal blood has not been assessed at present despite the fact that this peptide has been implicated in the control of several pituitary hormones and especially in the release of prolactin (PRL) caused by stress. In the present work the effect of stress on the in vivo release of VIP into the pituitary portal blood of male rats was evaluated by means of push-pull perfusion (PPP) of median eminence (EM). VIP content in the PPP liquid was successfully measured and their levels agree well with pituitary portal blood levels measured by other authors. Whereas plasma PRL levels strongly increased during acute immobilization (IMO), no changes in VIP secretion into the ME were observed. VIP release into the ME was also unaffected by exposure to ether. In contrast, in chronically immobilized rats a significant increase in VIP release into the ME was obtained in response to acute IMO. The present data argue against a major role of hypothalamic VIP in PRL release caused by acute stress and show that chronic stress qualitatively alters the signal of hypothalamic VIP to the pituitary.

Hypothalamic involvement in stress-induced hypocalcemia in rats

Although hormonal regulation of blood calcium homeostasis has been intensively investigated in the peripheral organs, the involvement of the central nervous system in calcium regulation is still poorly understood. In the present study, we found that (1) bilateral lesions of the ventromedial nucleus of the hypothalamus (VMH), but not those of the paraventricular hypothalamic nucleus or the lateral hypothalamic area, eliminated immobilization (IMB)-induced hypocalcemia, and (2) electrical stimulation of the VMH decreased the blood calcium level. The results suggest that the VMH has a hypocalcemic function and plays a role in IBM-induced hypocalcemia.

Immobilization stress-induced oral opioid self-administration and withdrawal in rats: role of conditioning factors and the effect of stress on "relapse" to opioid drugs.

The effect of 15 min/day of immobilization (IM) stress on oral self-administration (SA) of morphine (0.5 mg/ml) or fentanyl (25 micrograms/ml) and withdrawal was examined in rats. In addition, the role of conditioning factors in these effects was assessed. For each drug, four groups of subjects were exposed for 50 days to IM stress prior to the drug SA period [Paired-Stress (P-S) groups], to IM stress prior to the drug SA period on half of the days and after the drug SA period on the rest of the days [Partial Paired-Stress (PP-S) groups], to IM stress several hours after the drug SA period [Unpaired-Stress (UP-S) groups], or to no IM stress [Control (C) groups]. The P-S and PP-S groups increased their drug SA during choice days in which both the opioid solution and water were available, and tended to manifest a more severe withdrawal syndrome after a naloxone challenge compared with the UP-S and C groups. Reinstatement of the opioid SA under conditions of paired-stress or no stress was further examined after 3 weeks without exposure to either stress or drugs. The paired stress animals had higher levels of drug SA and manifested a more severe withdrawal syndrome than those tested without stress. These results indicate that the learned association between exposure to stress and the drug availability may mediate, in part, the stress-induced enhancement of opioid SA and withdrawal effects.

Effects of postoperative immobilization on the reconstructed anterior cruciate ligament. An experimental study in rabbits.

To investigate the effects of postoperative immobilization and limited motion on reconstructed anterior cruciate ligaments, 28 rabbits received an anterior cruciate ligament reconstruction using autogenous Achilles tendon and were then divided into three groups: fully immobilized, 4 weeks immobilized, and limited motion. Two rabbits from each group were evaluated macroscopically, histologically and microangiographically at 4-week intervals until 12 weeks postoperatively. An additional six rabbits in the 4 weeks immobilized and limited motion groups were studied biomechanically at 12 weeks postoperatively. Macroscopically, both immobilized groups showed more proliferation of the infrapatellar fat pad, which was adherent to the reconstructed anterior cruciate ligament. Histology revealed more rapid regeneration of reconstructed anterior cruciate ligaments in the limited motion group, with no findings of necrosis in the mid-substance. Microangiography indicated faster normalization of vascularity in the limited motion group. The biomechanical study showed no significant difference in laxity between the 4 weeks immobilized and limited motion groups. The graft stiffness and maximum load to failure were greater for the limited motion group, although the increase was not statistically significant. The histologic and microangiographic results from the limited number of animals in this study support limited postoperative motion in the anterior cruciate ligament reconstructed knee. However, there were no differences in terms of the biomechanical parameters at 12 weeks postoperatively between the immobilized and limited motion treatment modes.

Effects of single or repeated immobilization on release of norepinephrine and its metabolites in the central nucleus of the amygdala in conscious rats.

The release of norepinephrine (NE) and its metabolites in the central nucleus of the amygdala was measured using in vivo microdialysis during immobilization (IMMO) stress in conscious rats. Animals underwent 2-hour periods of IMMO either once or daily for 7 days. Extracellular fluid concentrations of NE, dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), and the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) were measured before, during, and after IMMO. Microdialysate levels of NE and DHPG attained 2- to 3-fold increments during the 1 h of IMMO and declined thereafter, whereas MHPG and DOPAC levels attained maximal levels of about twice basal concentrations during the 2- or 3-h after initiation of IMMO. After the sixth IMMO basal levels of NE, DHPG, MHPG, and DOPAC were decreased, and NE, DHPG, and DOPAC responses during the seventh IMMO failed to attain levels found during the first IMMO, although the absolute changes during IMMO were similar between animals subjected to IMMO once or seven times. The results indicate that acute IMMO increases synthesis, release, and metabolism of NE in the central nucleus of the amygdala and that repetition of IMMO decreases basal catecholamine synthesis and noradrenergic turnover in this brain region, without inhibiting acute noradrenergic responses.

Stress-induced norepinephrine release in the paraventricular nucleus of rats with brainstem hemisections: a microdialysis study.

Immobilization (IMMO) of conscious rats evokes marked increases in release of norepinephrine (NE) in the paraventricular nucleus (PVN) of the hypothalamus, consistent with a role of NE in the PVN release of corticotropin-releasing hormone and therefore in pituitary-adrenocortical activation during stress. The present study examined the effects of surgical hemisection of the brainstem between the locus ceruleus and rostral portion of the medulla on release of NE in the PVN of the hypothalamus in vivo in conscious rats, at baseline and during IMMO. Concentrations of NE, the intraneuronal NE metabolite dihydroxyphenylglycol (DHPG), and the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) were measured in microdialysate samples obtained beginning 24 h after implantation of a microdialysis probe in the PVN either ipsilateral or contralateral to the hemisection. On the lesioned side, baseline levels of NE, DHPG, and DOPAC were significantly lower and IMMO-induced increases were smaller than in sham-operated rats. Contralateral to the hemisection, DOPAC levels were significantly reduced. Neither baseline levels nor IMMO-induced increases in plasma corticosterone levels differed between lesioned and sham-operated animals. The present results indicate that: (1) NE release in the PVN at baseline and during IMMO depends mainly on ascending medullary tracts from ipsilateral brainstem A1 and A2 catecholaminergic areas, with small contributions from the locus ceruleus and from contralateral medullary cells, and (2) brainstem hemisection does not influence IMMO-induced activation of the hypothalamic-pituitary-adrenocortical axis as indicated by plasma corticosterone levels in conscious rats.

Evidence for the involvement of serotonin in acute stress-induced release of luteinizing hormone in the male rat

The influence of serotonin on luteinizing hormone (LH) release caused by exposure to two acute Stressors differing in their intensity (restraint in tubes and immobilization in woodboards) was studied in adult male rats. Inhibition of serotonin synthesis with p-chlorophenylalanine (PCPA) significantly abolished LH release caused by immobilization (IMO). Administration of the serotonin antagonists mianserine and methiothepin also eliminated LH release caused by IMO without altering basal LH levels. These data represent the first evidence that a classical neurotransmitter (serotonin) is involved in LH release caused by stress in the rat.

Effects of chronic immobilization stress on GH and TSH secretion in the rat: Response to hypothalamic regulatory factors

The effect of chronic immobilization (2 h/day) for 13 days on basal and stress levels of GH and TSH, and their response to various hypothalamic regulatory factors was studied in male Sprague-Dawley rats. Chronic immobilization (IMO) resulted in reduced serum TSH levels in stress situations but not in resting conditions. GH secretion was inhibited both in resting and stress situations. Chronic IMO impaired both GH and TSH responses to GRH and TRH, respectively, but also to another peptide (VIP) stimulatory for the two hormones. Whereas somatostatin administration inhibited GH secretion in control but not in chronic IMO rats, its inhibitory effect on TSH was slight and similar in the two experimental groups. The present results suggest that chronic exposure to a severe stressor such as IMO alters GH and TSH secretion, at least in part by changes in the response of the pituitary to the hypothalamic regulatory factors. The actual influence of chronic IMO on the release of these peptides into the median eminence remains to be studied.

Role of somatostatin in the acute immobilization stress-induced GH decrease in rat

In the present work we have investigated to what extent somatostatin (SRIF) release from median eminence (ME) is affected by stress immobilization (IMO) in unanesthetized rats stereotaxically implanted with a push-pull cannula (PPC). One week after implantation, the ME was perfused with artificial cerebrospinal fluid for 1 hour in basal, stress and recovery conditions respectively. Samples were collected every 15 min and SRIF was measured by RIA. In another group of animals, a jugular cannula was inserted the day before and plasma samples were taken off simultaneously with the ME perfusate for GH and SRIF analysis respectively. SRIF release from the ME is rapidly (15 min) and significantly increased (58 ± 11 vs 28 ± 5 pg/15 min; n=7; P < 0.01) in rats bearing only PPC. Intriguingly, animals bearing a jugular catheter plus a PPC showed no increase in SRIF release during the first 15 min of IMO in spite of a striking decrease of plasma GH (27.2 ± 3.8 vs 3.6 ± 1.3 ng/ml; n=6; P < 0.001) observed at this time. However, in spite that the animals responded with a significant increase in SRIF, the response was later and more reduced than in animals without jugular cannula. Since our two rat groups -as result of jugular cannula surgery 24 hours before- showed differences such as a food intake, body weight gain, plasma GH levels and basal SRIF release, we think that these differences could explain the modifications in the regulatory mechanisms involved in GH control under acute stress.

Evaluation of Extrication Orthoses in Immobilization of the Unstable Cervical Spine

Evaluation of Extrication Orthoses in Immobilization of the Unstable Cervical Spine