Serotonin effect on deiodinating activity in the rat

Serotonin (5-HT) and thyroid hormones are part of a complex system modulating eating behaviour and energy expenditure. 5-Deiodinase (5-D) converts the relatively inactive thyroxine (T4) to triiodothyronine (T3), and its activity is an indirect measure of T3 production in peripheral tissues, particularly in the brain, intrascapular brown adipose tissue (IBAT), heart, liver, and kidney. We evaluated the effect of 5-HT on 5'-D activity during basal conditions and after short (30 min) cold exposure (thyroid stimulating hormone stimulation test, TST). 5'-D activity was assessed in the liver, heart, brain, kidney, and IBAT. TST increases 5'-D activity in the brain, heart, and IBAT and decreases it in kidney, leaving it unchanged in the liver. 5-HT alone did not modify 5'-D activity in the organs under study but decreased it in the IBAT, heart, and brain when injected before the TST was administered. Our results confirm the important role of 5-HT in thermoregulation, given its peripheral site of action, in modulating heat production controlling intracellular T3 production. These effects are more evident when heat production is upregulated during cold exposure in organs containing type II 5'-D, such as the brain, heart, and IBAT, which are able to modify their function during conditions that alter energy balance. In conclusion, 5-HT may also act peripherally directly on the thyroid and organs containing type II 5'-D, thus controlling energy expenditure through heat production.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Food intake after hatching inhibits the growth hormone induced stimulation of the thyroxine to triiodothyronine conversion in the chicken.

The effect of a single injection of 10 micrograms chicken GH on circulating thyroid hormones as well as in vitro liver 5'-monodeiodination (5'-D) activity was studied in posthatch chicks submitted to different feeding conditions. One group was normally fed after hatching, a second group was only fed after three days and a third group was food deprived after 2 days of feeding. Combination of all results indicates that the start of food intake abolishes the stimulatory effect of a GH injection on circulating T3 and liver 5'-D activity. Food deprivation after a period of food intake restores the GH effect on plasma T3 but not on liver 5'-D.     

Induction of 5-Deiodinase Activity in Astroglial Cells by 12-O-Tetradecanoylphorbol 13-Acetate and Fibroblast Growth Factors

In the brain, 5'-deiodinase (5'-D) is responsible for the metabolic activation of thyroxine (T4) into 3,5,3'-triiodothyronine (T3) and 5-deiodinase (5-D) deiodinates T4 and T3 into inactive metabolites. This study examines the effects of factors known to induce astroglial 5'-D activity on the 5-D activity in cultured rat astroglial cells. The potencies of these factors were compared after 8 h of incubation, when stimulations by these factors near their maximal effects. 12-O-Tetradecanoylphorbol 13-acetate (TPA) at 10(-7) M was a potent inducer of 5-D activity, producing a 30- to 80-fold increase after 8 h. The maximal effect of TPA was observed after about 14 h. The TPA stimulation of 5-D activity was not dependent on glucocorticoids, unlike 5'-D activity. In comparison with TPA, 8-bromo-cyclic AMP (10(-3) M) was a poor inducer of 5-D activity whereas it is an excellent inducer of 5'-D activity. It produced a 2- to 20-fold increase in 5-D activity after 8 h. Natural acidic fibroblast growth factor (20 ng/ml) produced a degree of stimulation similar to that of TPA after 8 h. The maximal effect of acidic fibroblast growth factor was observed after about 16 h (until a 120-fold increase). Recombinant acidic fibroblast growth factor also induced 5-D activity. Basic fibroblast growth factor was less potent than acidic fibroblast growth factor for increasing 5-D activity (maximal increase by 40- to 50-fold after 8 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Na-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid percussion injury

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). An important early component of the edema associated with TBI is astrocyte swelling (cytotoxic edema). Mechanisms for such swelling, however, are poorly understood. Ion channels/transporters/exchangers play a major role in cell volume regulation, and a disturbance in one or more of these systems may result in cell swelling. To examine potential mechanisms in TBI-mediated brain edema, we employed a fluid percussion model of in vitro barotrauma and examined the role of the ion transporter Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) in trauma-induced astrocyte swelling as this transporter has been strongly implicated in the mechanism of cell swelling in various neurological conditions. Cultures exposed to trauma (3, 4, 5 atm pressure) caused a significant increase in NKCC1 activity (21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure, trauma significantly increased NKCC1 activity at 1 h and it remained increased for up to 3 h. Trauma also increased the phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition of MAPKs and oxidative/nitrosative stress diminished the trauma-induced NKCC1 phosphorylation as well as its activity. Bumetanide, an inhibitor of NKCC1, significantly reduced the trauma-induced astrocyte swelling (61%). Silencing NKCC1 with siRNA led to a reduction in trauma-induced NKCC1 activity as well as in cell swelling. These findings demonstrate the critical involvement of NKCC1 in the astrocyte swelling following in vitro trauma, and suggest that blocking NKCC1 activity may represent a useful therapeutic strategy for the cytotoxic brain edema associated with the early phase of TBI.     

Avian hepatic T-3 production by two pathways of 5'-monodeiodination: effects of fasting and patterns during development

Two forms of iodothyronine 5'-monodeiodinase (5'-D) were studied in liver homogenates from adult and developing quail. The influence of fasting in adults and corticosterone treatment in embryonic quail on 5'-D also were examined. Liver homogenates were assayed for 5'-D activity in the presence of abundant substrate (T4) and cofactor (dithiothreitol; DTT). Generation of T3 during a 15 min incubation at 37 degrees C was assessed by an ethanol-based RIA. In adults, both Type I [the fraction of activity inhibited by propylthiouracil (PTU)] and a putative Type II (the PTU-insensitive fraction) were present in liver homogenates. Type II activity typically comprised about 30% of Total activity. Type I activity first appeared on day 15 of the 16.5 day incubation period, increased 20-fold to peak at hatching, then gradually declined to reach adult levels by 21 days of age. Type II activity was present at all developmental stages and was highest during the perinatal period. Corticosterone treatment in vivo on day 13 of development induced increases in both Type I and Type II activities in liver homogenates 24- and 48-h after treatment. This study demonstrates that in avian liver a putative Type II 5'-D activity (generally considered to be lacking in mammalian liver) is present and may be important in the maintenance of minimal concentrations of tissue T3 during fasting. Both types of 5'-D contribute to the developmental pattern of serum T3 concentrations. Type II comprises a large proportion of total activity during late embryonic life; Type I becomes predominant at the beginning of the perinatal period.(ABSTRACT TRUNCATED AT 250 WORDS)     

SPARC/Osteonectin mRNA Is Induced in Blood Vessels Following Injury to the Adult Rat Cerebral Cortex

Recently we described the pattern of expression of the anti-adhesive glycoprotein SPARC/osteonectin in the developing and adult brain. SPARC mRNA was present in developing blood vessels during neurogenesis, but was not detected in the mature vasculature. We have now examined the effect of a lesion to the adult rat cerebral cortex on the expression of SPARC by in situ hybridization. SPARC mRNA was increased in the zone proximal to the wound at 3 to 10 days after cortical brain injury. During this period, SPARC was induced in mature blood vessels close to the lesion site and in blood vessels which develop following injury. These results suggest a role for SPARC in the process of angiogenesis following injury to the adult cerebral cortex.     

一种有效区分移植细胞和宿主细胞脑损伤模型的建立

为了区分移植神经细胞和宿主细胞,便于将来在宿主体内对移植细胞进行在体的电生理记录以及其它方面的研究,通过机械损毁的方法,建立了一种特殊的脑损伤模型。结果发现,通过机械损毁的方法,在大鼠大脑皮层形成形态规则的损伤空洞,其模型稳定,重复性好;在空洞内进行干细胞移植,能够长时间存活,移植神经干细胞绝大部分细胞分化为神经元,只有少量细胞分化为胶质细胞,而且移植细胞与宿主细胞分界明显;对移植细胞进行单细胞电生理记录,记录到神经元放电信号。这些结果表明,通过机械损毁的方法,在大鼠大脑皮层成功建立了一个稳定、精确定位移植细胞与宿主细胞界限的脑损伤模型。     

Changes in autophagy proteins in a rat model of controlled cortical impact induced brain injury

Autophagy has been implicated in several neurodegenerative diseases and recently its role in acute brain injury has received increased interest. In our study, we investigated the profiles of autophagy-linked proteins (MAP-LC3 (Atg8), beclin-1 (Atg6) and the beclin-1-binding protein, bcl-2, following controlled cortical impact injury in rats—a model for moderate-to-severe traumatic brain injury. We observed significant increases in the levels of the processed form of LC3 (LC3-II) in the ipsilateral cortex 2 h to 2 days after injury when compared to sham. Furthermore, the beclin-1/bcl-2 ratio in the ipsilateral cortex was found to have increased from 1 and 2 days after injury. Since both of these changes are established autophagy-enabling events, and, based on these data, we propose that autophagy, plays a role in the manifestation of cell injury following brain trauma.     

Evidence for chicken GH as the only hypophyseal factor responsible for the stimulation of hepatic 5'-monodeiodination activity in the chick embryo

The influence of an intravenous injection of chicken growth hormone (cGH), a total chicken pars distalis (PD) extract, and a PD extract depleted of cGH by immunoadsorption was studied in the 18-d-old chick embryo. Plasma concentrations of triiodothyronine (T3), thyroxine (T4), and hepatic 5'-monodeiodination (5'-D) activity were measured. An injection of total PD extract raised plasma T3, T4, and 5'-D activity, whereas a PD extract depleted of GH only increased plasma T4. The amount of cGH present in the PD extracts, as measured by homologous cGH radioimmunoassay, increased T3 and raised liver 5'-D, but had no effect on plasma T4. The effect on liver 5'-D was more pronounced with cGH than with a total PD extract, whereas the effect on plasma T3 was somewhat less pronounced. It was concluded that cGH increased the peripheral conversion of T4 into T3 in the chick embryo, whereas a PD extract depleted of cGH was purely thyrotropic. The PD extract also seemed to have 5'-D-suppressing activity.     

Effects of short-term exposure to lithium on antiapoptotic Bcl-xL protein expression in cortex and hippocampus of rats after acute stress

The antiapoptotic protein Bcl-xL is involved in development of neurobiological resilience to stress; hence, the possibility of use of psychotropic drugs to increase its expression in brain in response to stress is of considerable interest. Lithium is a neurotropic drug widely used in psychiatry. In work, we studied effects of lithium administration (for 2 or 7 days) on the expression of Bcl-xL mRNA and protein in the hippocampi and cortices of rats subjected to stress that induced depression-like behavior in the animals. In contrast to the brain-derived neurotrophic factor (BDNF), whose expression decreased in the hippocampus in response to acute stress, stress increased the level of Bcl-xL mRNA in the hippocampus, but decreased it in the frontal cortex. Treatment of stressed animals with lithium for 2 or 7 days increased Bcl-xL protein levels 1.5-fold in the hippocampus, but it decreased them in the cortex. Therefore, Bcl-xL expression in the brain can be modulated by both stress and psychotropic drugs, and the effects of these factors are brain region-specific: both stress exposure and lithium administration activated Bcl-xL expression in the hippocampus and suppressed it in the frontal cortex. The activation of Bcl-xL expression in the hippocampus by lithium, demonstrated for the first time in this study, suggests an important role of this protein in the therapeutic effects of lithium in the treatment of stress-induced psychoemotional disorders.     

Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.     

Differential responses of rat pineal thyroxine type II 5′-deiodinase andN-acetyltransferase activities to either light exposure,isoproterenol, phenylephrine,or propranolol

1. Compared to pineal N-acetyl transferase (NAT) activity, which exhibited a dramatic drop following acute light exposure at night, nocturnal rat pineal thyroxine type II 5'-deiodinase (5'-D) activity was minimally influenced by the same light exposure. The injection of cycloheximide, a potent inhibitor of protein synthesis, although it did curtail the rise in NAT activity for at least 2 hr, did not elicit decreases in the activities of either 5'-D or NAT enzymes. Propranolol, a beta-adrenergic blocker, either delayed the continued nocturnal rise in 5'-D activity when injected at 0000 hr or slightly enhanced the fall in 5'-D activity when injected at 0200 hr. These results suggest that interruption of the synthesis of proteins is responsible for the slow deterioration of 5'-D activity induced by either light or propranolol. 2. The slight fall in 5'-D activity induced by light at night was prevented by isoproterenol; phenylephrine, however, did not prevent the fall and the effect of isoproterenol + phenylephrine was similar to that obtained with isoproterenol alone. On the other hand, the light-inhibited NAT activity recovered after the injection of isoproterenol; phenylephrine did not elicit any effect, but the injection of both isoproterenol and phenylephrine simultaneously caused a greater NAT response than that induced by isoproterenol alone. 3. When injected during the day, phenylephrine had no effect on either pineal 5'-D or NAT activities; however, the injection of either isoproterenol alone or isoproterenol + phenylephrine elicited 5-fold and 10-fold increases in nocturnal, light-suppressed 5'-D and NAT activities, respectively. During the day, phenylephrine did not potentiate the effects of isoproterenol on NAT activity as it did at night. When the effects of isoproterenol on the 5'-D activity were compared to rats exposed to light during the day and at night, the activity of 5'-D reached a higher level at night than during the day.     

In vivo stimulation of rat pineal type II thyroxine 5'-deiodinase activity by either norepinephrine or isoproterenol

Herein we show, for the first time, a very marked increase in thyroxine 5'-deiodinase (5'-D) activity in rats injected with norepinephrine (NE) and desmethylimipramine, a drug which inhibits NE uptake by nerve terminals. The response to NE was greater in pineals collected from hypothyroid animals than in glands from euthyroid animals. NE was more effective in stimulating pineal 5'-D than was isoproterenol, suggesting that, in addition to beta-adrenergic receptors, alpha-adrenergic receptors might be involved in the 5'-D activation. However, phenylephrine, an alpha-adrenergic agonist, did not potentiate the effect of isoproterenol on pineal 5'-D activity. The nocturnal increase in pineal 5'-D activity was completely abolished by propranolol, a beta-adrenergic receptor blocker, while prazosin, an alpha-adrenergic receptor blocker, had minimal effect. These results show that the role of alpha-receptors in promoting the NE-mediated rise in rat pineal 5'-D activity is minor in contrast to the role of beta-adrenergic receptors.     

Effects of Bradykinin Postconditioning on Endogenous Antioxidant Enzyme Activity After Transient Forebrain Ischemia in Rat

Bradykinin is considered an important mediator of the inflammatory response in both the peripheral and the central nervous system and it has attracted recent interest as a potential mediator of brain injury following stroke. Bradykinin is recognized to play an important role in ischemic brain. We investigated the effect of bradykinin postconditioning on ischemic damage after 8 min of ischemia (four-vessel occlusion) and 3 days of reperfusion. Bradykinin was administered after 2 days of reperfusion at a dose of 150 μg/kg (i.p.). Catalase (CAT) activity was significantly increased in all examined regions (cortex, hippocampus and striatum) 3 days after 8 min of ischemia, but postconditioning decreased this activity below the control values. The total activity of superoxide dismutase (SOD) 3 days after ischemia was at control level with or without postconditioning. However, the analysis of individual SODs separately revealed interesting differences; while the activity of CuZnSOD was significantly decreased 3 days after ischemia, the activity of MnSOD was significantly increased compared to control levels. In both cases, postconditioning returned SOD activity to control levels. These findings are interesting because MnSOD is a mitochondrial enzyme and its activity in the cytosol suggests that a possible mechanism of protection provided by postconditioning could include prevention of release of mitochondrial proteins to the cytoplasm, resulting in protection against the mitochondrial pathway of apoptosis. 8 min of ischemia alone caused the degeneration of 52.37% neurons in the hippocampal CA1 region 3 days later. Bradykinin used as postconditioning 2 days after the same interval of ischemia enabled the survival of more than 97% of CA1 neurons. This study demonstrated that bradykinin postconditioning induces protection against ischemic brain injury and promotes neuronal survival.     

-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (_N = 15.4 and ^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of -phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.