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Neonatal hypoxic-ischemic (HI) brain injury causes severe brain damage in newborns. Following HI injury, rapidly accumulating oxidants injure neurons and interrupt ongoing developmental processes. The antioxidant, sodium pyruvate, has been shown to reduce neuronal injury in neonatal rats under conditions of oxygen glucose deprivation (OGD) and HI injury. In this study, we evaluated the effects of ethyl pyruvate (EP) and insulin-like growth factor-I (IGF-I) alone or in combination in a similar setting. For this purpose, we used an in vitro model involving primary neonatal rat cortical neurons subjected to OGD for 2.5 h and an in vivo model involving unilateral carotid ligation in rats on post-natal day 7 with exposure to 8% hypoxia for 2.5 h. The cultured neurons were examined by lactate dehydrogenase (LDH) and cell viability assays. For the in vivo experiments, behavioral development was evaluated by the foot fault test at 4 weeks of recovery. 2,3,5-Triphenyltetrazolium chloride monohydrate and cresyl violet staining were used to evaluate HI injury. The injured neurons were Fluoro-Jade B-labeled, new neuroprecursors were double labeled with bromodeoxyuridine (BrdU) and doublecortin, new mature neurons were BrdU-labeled and neuronal nuclei were labeled by immunofluorescence. Under conditions of OGD, the LDH levels increased and neuronal viability decreased. Treatment with 0.5 mM EP or 25 ng/ml IGF-I protected the neurons (P

Background Neonatal hypoxia-ischemia (HI) remains a major cause of severe brain damage and is often associated with high mortality and lifelong disability. Immature brains are extremely sensitive to hypoxia-ischemia, shown as prolonged mitochondrial neuronal death. Sodium pyruvate (SP), a substrate of the tricarboxylic acid cycle and an extracellular antioxidant, has been considered as a potential treatment for hypoxic-ischemic encephalopathy (HIE), but its effects have not been evaluated in appropriate animal models for hypoxic-ischemic encephalopathy (HIE). Methods This investigation employed primary cortical neuron cultures derived from neonatal rats subjected to oxygen and glucose deprivation (OGD) and a well-established neonatal rat hypoxia-ischemia model. Results HI caused brain tissue loss and impaired sensorimotor function and spatial memory while SP significantly reduced brain damage and improved neurological performance. These neuroprotective effects of SP are likely the result of improved cerebral metabolism as demonstrated by maintaining ATP levels and preventing an increase in intracellular reactive oxygen species (ROS) levels. SP treatment also decreased levels of Bax, a death signal for immature neurons, blocked caspases-3 activation, and activated a key survival signaling kinase, Akt, both in vitro and in vivo. Conclusion SP protected neonatal brain from hypoxic-ischemic injury through maintaining cerebral metabolism and mitochondrial function.

Aims: Studies employing transgenic mice indicate that overexpression of superoxide dismutase 1 (SOD1) improves memory during aging. It is unclear whether the improvement is due to a lifetime of overexpression, decreasing the accumulation of oxidized molecules, or if increasing antioxidant enzymes in older animals could reduce oxidative damage and improve cognitive function. We used adeno-associated virus to deliver antioxidant enzymes (SOD1, SOD2, catalase [CAT], and SOD1+CAT) to the hippocampus of young (4 months) and aged (19 months) F344/BN F1 male rats and examined memory-related behavioral performance 1 month and 4 months postinjection. Results: Overexpression of antioxidant enzymes reduced oxidative damage; however, memory function was not related to the level of oxidative damage. Increased expression of SOD1, initiated in advanced age, impaired learning. Increased expression of SOD1+CAT provided protection from impairments associated with overexpression of SOD1 alone and appears to guard against cognitive impairments in advanced age. Innovation: Viral vector gene delivery provides a novel approach to test the hypothesis that increased expression of antioxidant enzymes, specifically in hippocampal neurons, will provide protection from age-related cognitive decline. Further, expression of multiple vectors permits more detailed investigation of mechanistic pathways. Conclusion: Oxidative stress is a likely component of aging; however, it is unclear whether increased production of reactive oxygen species or the accumulation of oxidative damage is the primary cause of functional decline. The results provide support for the idea that altered redox-sensitive signaling rather than the accumulation of damage may be of greater significance in the emergence of age-related learning and memory deficits. Antioxid. Redox Signal. 16, 339–350.

Hypoxia preconditioning and subsequent tolerance to hypoxia-ischemia damage is a well-known phenomenon and has significant implications in clinical medicine. In this investigation, we tested the hypothesis that the transcriptional activation of IGF-I is one of the underlying mechanisms for hypoxia-induced neuroprotection. In a rodent model of hypoxia-ischemia, hypoxia preconditioning improved neuronal survival as demonstrated by decreased hypoxia-ischemia-induced neuronal apoptosis. To study the role of IGF-I in hypoxia tolerance, we used in situ hybridization to examine IGF-I mRNA distribution on adjacent tissue sections. In cerebral cortex and hippocampus, hypoxia preconditioning resulted in an increase in neuronal IGF-I mRNA levels with or without hypoxia-ischemia. To test its direct effects, we added IGF-I to primary neuronal culture under varying oxygen concentrations. As oxygen concentration decreased, neuronal survival also decreased, which could be reversed by IGF-I, especially at the lowest oxygen concentration. Interestingly, IGF-I treatment resulted in an activation of hypoxia-inducible factor 1alpha (HIF-1alpha), a master transcription factor for hypoxia-induced metabolic adaptation. To evaluate whether IGF-I transcriptional activation correlates with HIF-1alpha activity, we studied the time course of HIF-1alpha DNA binding activity in the same rat model of hypoxia-ischemia. After hypoxia-ischemia, there was an increase in HIF-1alpha DNA binding activity in cortical tissues, with the highest increase around 24 h. Like IGF-I mRNA levels, hypoxia preconditioning increased HIF-1alpha DNA binding activity alone or with subsequent hypoxia ischemia. Overall, our results suggest that IGF-I transcriptional activation is one of the metabolic adaptive responses to hypoxia, which is likely mediated by a direct activation of HIF-1alpha.

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy- d - [1- 14 C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1 −/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy- d - [1- 14 C]glucose is significantly reduced in synaptosomes prepared from Igf1 −/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1 −/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in Igf1 −/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.

In adults, insulin-like growth factor binding protein 3 (IGFBP3) is the main carrier protein for circulating insulin-like growth factors (IGFs) (IGF-I and -II). While most IGFBP3 is synthesized in the liver, it is also expressed locally by many cell types including vascular endothelial cells. The regulation of this endothelial IGFBP3 expression, especially in response to hypoxic-ischemic injury, has not been investigated in vivo. Using in situ hybridization histochemistry, we studied the cellular distribution of IGFBP3 mRNA in rat brains following hypoxic-ischemic injury at 1, 5, 24, and 72 h of recovery. In normal P7 rat brain, IGFBP3 mRNA was found in neurons within the thalamus, hippocampus, and amygdaloid. Low levels of IGFBP3 mRNA were also detected in cerebral vascular endothelial cells. After the hypoxic-ischemic injury, the levels of neuronal IGFBP3 mRNA substantially decreased within 24 h in areas that were normally supplied by the middle cerebral artery. In the meantime, there was an immediate increase in IGFBP3 expression in vascular endothelial cells throughout the affected hemisphere. This vascular IGFBP3 expression was further enhanced with the highest level at 24 h of recovery whereas neuronal IGFBP3 expression was further decreased. By 72 h of recovery, IGFBP3 was no longer expressed in vascular endothelial cells. Taken together, the activation of IGFBP3 is a likely mechanism by which vascular endothelial cells respond to hypoxic-ischemic insult. In addition, increased endothelial IGFBP3 may modulate the interaction of IGFs with IGF-I receptors at the site of injury and/or act independently on endothelial cell growth.

Hypoxia-ischemia induces apoptotic and necrotic cell death, which results partially from persistent alterations in cellular energy homeostasis. Insulin-like growth factor I (IGF-I) is an anabolic pleiotrophic factor required by developing neurons for their optimal proliferation, differentiation, and survival. To determine how cell death and changes in IGF-I gene expression relate to the extent of hypoxia-ischemia, we evaluated the time course of apoptosis in a neonatal hypoxia-ischemia model in relation to the cellular distribution of IGF-I and IGFBP5 mRNA. Severe hypoxia-ischemia results in an immediate decrease in neuronal IGF-I and IGFBP5 mRNA. The decrease in neuronal IGF-I mRNA was concurrent with an increase in the number of apoptotic cells. It is conceivable that the immediate decrease in IGF-I gene expression may contribute to the impending neuronal death and selective vulnerability of myelinogenesis during the perinatal period.

The brain damage produced by unilateral cerebral hypoxia-ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood-brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia-ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic-ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia-ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.

Two genes whose expression is likely to be altered during diabetes mellitus are aldose reductase (AD) and insulin-like growth factor-I (IGF-I). We proposed that gene expression of AD is increased in vascular smooth muscle during diabetes mellitus due to hyperglycemia, while IGF-I expression is decreased in insulin-deficient diabetes and elevated in insulin-resistant diabetes. The mRNA for both was measured in the renal glomerulus, in the vascular smooth muscle of large arterioles from the brain, kidney, and small intestine, and in the aorta of hypoinsulinemic streptozotocin (STZ)-treated rats and hyperinsulinemic Zucker diabetic fatty (ZDF) rats. Quantitative in situ hybridization was used to determine variations in expression. Expression of the AD gene was unchanged in STZ and ZDF rats, except for a decrease of about 50% in glomeruli and renal smooth muscle of STZ diabetic rats. Expression of IGF-I generally decreased in vascular smooth muscle of insulin-depleted STZ diabetic rats, but was normal in hyperinsulinemic ZDF rats. The data indicate that decreased expression of the AD gene is a specific problem in renal vascular smooth muscle and glomeruli in the insulin-depleted STZ model of diabetes. The expression of the IGF-I gene in vascular muscle was decreased in hypoinsulinemic diabetic animals, but did not increase in hyperinsulinemic diabetic rats.

Insulin-like growth factor I (IGF-I) is an anabolic pleiotrophic factor essential for postnatal rat brain development, especially during the first 21 days, the “critical growth period.” Cerebral hypoxic–ischemic insults occurring during the perinatal period can result in neuronal necrosis and permanent brain damage. To understand the regulation of the action of IGF-I in response to such a metabolic insult, we investigated the gene expression of IGF-I, type I IGF receptor, IGF binding protein (IGFBP) 2, and IGFBP5 during the first 72 h after hypoxia–ischemiain the immature rat. At 1 h of recovery, messenger RNA (mRNA) levels of all IGF system components were decreased throughout the hemisphere ipsilateral to the carotid artery ligation. This decrease is more pronounced at 24 h of recovery, especially in areas vulnerable to hypoxic–ischemic injury, such as the thalamus and hippocampus. At 72 h of recovery, although IGFBP2 and type 1 IGF receptor mRNA levels remain suppressed, gene expression of both IGF-I and IGFBP5 was activated in reactive astrocytes. Therefore, during the critical growth period in rats, the transcriptional levels of all IGF system components are extremely sensitive to metabolic perturbations associated with cerebral hypoxia–ischemia. The immediate decrease in IGF-I gene expression may be partially responsible for the impending neuronal death and selective vulnerability of myelinogenesis during the perinatal period.

To explore the protective effects of insulin-like growth factor-1(IGF-1) on the survival and apoptosis of cortical neurons of neonatal rat under oxidative stress and its significance.Primary cortical neurons from newborn rat were cultured and the oxidative stress model was established. Then cells were randomly divided into IGF-1 group and control group. The concentration of LDH in supernatant was detected. Cell survival was determined with MTT assay and the expression of active Caspase-3 was measured using Western Blotting.(1) The values of LDH gradually decreased with the increasing IGF-1 added to the cells [(0.5065 ± 0.0064) to (0.435 ± 0.0065), (P0.01)], but when the concentration of IGF-1 reached a certain level (25 ng/ml), there were no longer obvious effects on the level of LDH [(0.42 ± 0.012) to (0.418 ± 0.0098), (P0.05)]; Western blot showed that the level of active Caspase-3 was significantly decreased after treatment with IGF-1 [(0.662 ± 0.033) to (0.199 ± 0.01), (P0.01)]. (2) Compared with control group, without or with low concentration of H2O2 (0 - 40 µM), the values of LDH and the expression of active Caspase-3 in IGF-1 group were significantly decreased[(1.518 ± 0.137) to (1.068 ± 0.067), (P0.05) and 0.850 ± 0.042 to 0.597 ± 0.03, P0.01, respectively] while the values of MTT obviously elevated [(0.773 ± 0.062) to (1.196 ± 0.057), (P0.05)]; but with higher concentration (≥ 60 µM) of H2O2, the values of LDH and MTT and the expression of active Caspase-3 in IGF-1 group all had no significant difference (P0.05). (3) When the concentration of H2O2 reached 60 µM and higher, whatever concentration of IGF-1 could not lower the level of LDH compared with control group [(2.376 ± 0.04) to (2.442 ± 0.046), (P0.05)].Oxidative stress can induce IGF-1 resistance of cortical neurons in neonatal rat, and even increasing the concentration of IGF-1 can not restore their sensitivity to IGF-1.

Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca ( 2+) channels. Building on our discovery of the interaction and regulation of Ca ( 2+) channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca ( 2+) channel. Remarkably, we also found that this region attenuated Ca ( 2+) influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2's role in excitotoxicity and neuroprotection.

Maternal ethanol (ETOH) exposure is associated with impaired fetal growth. Because insulin-like growth factors (IGFs) are thought to be important in the regulation of fetal somatic growth, we examined the influence of maternal ETOH exposure on fetal growth and plasma levels of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in the rat model. Control (A) dams were fed a standard rat chow ad libitum. ETOH (E) consuming dams were fed a 36% ETOH diet, and pair-fed (P) dams were fed isocaloric amounts of a control liquid diet. All animals were killed on day 20 of gestation. Plasma concentrations of IGF-I and -II were determined by radioimmunoassay after formic acid-acetone extraction and heat inactivation of IGFBPs. Levels of IGFBPs in fetal plasma were estimated by Western ligand blotting after protein separation by SDS-PAGE and electrotransfer to nitrocellulose. Membranes were probed with [125l]IGF-l, and IGFBPs were identified by autoradiography, quantified by scanning densitometry and results expressed relative to corresponding IGFBPs in control fetal plasma. Maternal weight gain from conception to 20 days of pregnancy was reduced for E compared to P and A dams (p < 0.05 E vs. P or A). The same pattern was reflected in fetal weight that tended to be lower in P compared with A pups, and was significantly reduced in E pups compared with both groups (p < 0.0001 E vs. P or A). Thus, fetal growth was more retarded in E animals despite equal caloric and protein intake by E and P dams. Radioimmunoassay of fetal plasma revealed that there was no difference in circulating levels of IGF-II in A (65.5 ± 8.7 ng/ml) and P (65.9 ± 9.8 ng/ml) plasma. However, levels of IGF-II were higher in E (87.1 ± 6.2 ng/ml) than in corresponding P animals. At the same time, there were no differences in IGF-I levels between any of the treatment groups. Western ligand blotting demonstrated that levels of 32–34 kDa IGFBPs were elevated in plasma of P compared with A fetal plasma, consistent with an effect of reduced maternal nutrition. In contrast, levels of32–34 kDa IGFBPs were reduced in E plasma compared with P and A (p < 0.04 and p < 0.02, respectively). These data suggest that circulating levels of IGF-II (but not IGF-I) and specific IGFBPs are altered in growth-retarded fetuses exposed to ETOH. Comparison to fetal plasma obtained from pair-fed litters demonstrate that these effects on IGF and IGFBP levels are specific to ETOH, and not simply due to maternal nutrient intake. Taken together, these observations indicate that ETOH has unique effects on fetal IGF physiology compared with previously studied models of fetal growth retardation. Alterations in the expression and levels of IGFs and IGFBPs may contribute to ETOH-induced fetal growth deficiency.

Insulin-like growth factor binding protein-2 binds insulin-like growth factors I and II with high affinity and modulates the interaction of these ligands with the type I insulin-like growth factor receptor. Previously we have shown that insulin-like growth factor binding protein-2 and insulin-like growth factor-I gene expression are spatiotemporally co-ordinated in the developing retina and cerebellum. The present study examined other brain regions and found a similar correlation in insulin-like growth factor binding protein-2 and insulin-like growth factor-I gene expression in relay stations of developing sensory and cerebellar networks of the rat. In these sites, as in the cerebellum and retina, insulin-like growth factor-I messenger RNA is localized in the principal or projection neurons and insulin-like growth factor binding protein-2 messenger RNA is localized in surrounding astroglia. Outside these sensory relay centers, the relationship of insulin-like growth factor binding protein-2 to insulin-like growth factor-I gene expression is not so well defined. In the hippocampal formation, insulin-like growth factor-I messenger RNA is present in large interneurons and insulin-like growth factor binding protein-2 messenger RNA in regional astrocytes; their timing is co-ordinated, with peak levels seen about postnatal day 12, but their anatomical association is not apparent. The least degree of correlation between local insulin-like growth factor-I and insulin-like growth factor binding protein-2 gene expression is found in the neocortex, where insulin-like growth factor-I is abundant in scattered large neurons from postnatal days 3 to 20. In contrast, insulin-like growth factor binding protein-2 messenger RNA is widely expressed throughout the neocortex from before birth to about postnatal day 12, in a pattern consistent with expression by nascent astroglia. Insulin-like growth factor binding protein-2 gene expression is greatly reduced throughout the brain by the third week after birth; in response to optic nerve transection, however, there is a resurgence of gene expression for this factor by activated astrocytes in affected retinal target regions. Insulin-like growth factor binding protein-2 and insulin-like growth factor-II messenger RNAs are co-localized in the choroid plexus and leptomeninges from the time of birth onward without diminution. In summary, insulin-like growth factor binding protein-2 demonstrates complex patterns of gene expression during postnatal brain development—some of which appear to be closely related to local insulin-like growth factor synthesis and some of which appear independent of it. The spatiotemporal correlation between astroglial insulin-like growth factor binding protein-2 and neuronal insulin-like growth factor-I expression is restricted to maturing sensory and cerebellar projection systems, suggesting a system-specific function for insulin-like growth factor-I and insulin-like growth factor binding protein-2 interaction in the differentiation of these centers.

To evaluate the effects of estrogen on the rat pituitary insulin-like growth factor (IGF) system, binding of [125I]IGF-I and in situ hybridization for IGF-I, the IGF-I receptor and IGF binding protein-2 (IGFBP-2) were coupled with quantitative autoradiography. The groups included intact cycling females, intact males, and gonadectomized males and females with or without estrogen pellet implants. Binding of [125I]IGF-I in the anterior lobe of the pituitary occurred in dense clusters over a diffuse lower density background. [125I]IGF-I binding was significantly increased in the estrogen-treated groups and was highest at proestrus compared to the rest of the estrous cycle. IGF-I receptor messenger RNA (mRNA) was distributed diffusely through the anterior pituitary and was not different between the respective gonadectomized and estrogen-treated groups. IGFBP-2 mRNA was clustered throughout the anterior pituitary and was significantly higher in the estrogen-treated groups as noted above for [125I]IGF-I binding. IGF-I mRNA was diffuse throughout the anterior pituitary and was also significantly higher in the estrogen-treated groups. In the neural lobe, [125I]IGF-I binding, IGFBP-2 mRNA, IGF-I receptor mRNA, and IGF-I mRNA were all uniformly distributed and did not differ between groups. The results show that circulating estrogen differentially regulates components of the pituitary IGF-I system in a region-specific manner and suggest that a portion of IGF binding in the anterior pituitary may be to IGFBP-2.

Peripheral nerves from aged animals exhibit features of degeneration, including marked fiber loss, morphological irregularities in myelinated axons and notable reduction in the expression of myelin proteins. To investigate how protein homeostatic mechanisms change with age within the peripheral nervous system, we isolated Schwann cells from the sciatic nerves of young and old rats. The responsiveness of cells from aged nerves to stress stimuli is weakened, which in part may account for the observed age-associated alterations in glial and axonal proteins in vivo. Although calorie restriction is known to slow the aging process in the central nervous system, its influence on peripheral nerves has not been investigated in detail. To determine if dietary restriction is beneficial for peripheral nerve health and glial function, we studied sciatic nerves from rats of four distinct ages (8, 18, 29 and 38 months) kept on an ad libitum (AL) or a 40% calorie restricted diet. Age-associated reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated Schwann cell phenotype. The improvements in nerve architecture with diet restriction, in part, are underlined by sustained expression of protein chaperones and markers of the autophagy-lysosomal pathway. Together, the in vitro and in vivo results suggest that there might be an age-limit by which dietary intervention needs to be initiated to elicit a beneficial response on peripheral nerve health.

The measurement of serum insulin-like growth factors (IGFs) in serum is complicated by the presence of high affinity IGF-binding proteins. The accurate measurement of IGFs by radioligand binding assays requires that the interference from binding proteins be eliminated. Acid-gel chromatography, the standard method for removing binding proteins, is laborious and time consuming. Alternative methods for extracting serum IGFs include the use of HCl-ethanol treatment and reverse phase minicolumns. However, these methods are unsuitable for use with serum for some species, such as rat and sheep, due to incomplete removal of binding proteins. We developed a fast protein liquid chromatography size-exclusion chromatographic method for characterizing the presence of IGF-binding proteins in physiological fluids and used this method to systematically investigate different combinations of acids and organic solvents as potential extraction methods for IGFs. We developed and validated an improved extraction procedure that uses formic acid, Tween-20, and acetone. The new extraction method was used in conjunction with purified biosynthetic human IGF-II and a commercially available anti-IGF-II monoclonal antibody in the development of an improved RIA for IGF-II. The new RIA is sensitive (5.0 pg/tube), specific (IGF-I cross-reactivity, less than 1%), and reproducible [interassay precision (coefficient of variation), less than 9.2%). We measured the serum concentrations of IGF-II in adults and found a significant difference between normal subjects and individuals with insulin-dependent diabetes mellitus.

Oxidative stress-induced neuronal death due to hydrogen peroxide overload plays a critical role in the pathogenesis of numerous neurological diseases. Insulin-like growth factor-1 (IGF-1) is important in maintaining neuronal survival, proliferation, and differentiation in the central nervous system. We now report that sublethal doses of hydrogen peroxide attenuated IGF-1 neuroprotective activity on cultured cerebellar granule neurons under potassium and serum deprivation. Interestingly, this attenuation can be prevented by minocycline, an antibiotic that has been shown to have neuroprotective activity in animal models of neuronal injury. Furthermore, hydrogen peroxide also blocked IGF-1's neuroprotection for cortical neurons deprived of neurotrophic factors (B27), which was prevented by minocycline. Our data suggest that inhibition of IGF-1 signaling by hydrogen peroxide may constitute an additional pathway contributing to its neurotoxicity. More importantly, combining minocycline and IGF-1 could be an effective treatment in neurological diseases associated with both oxidative stress and deficiency of IGF-1.

Aged (20-22 months) male Fischer 344 rats were randomly assigned to sedentary (A-SED), environmentally-enriched (A-ENR), or exercise (A-EX) conditions. After 10-12 weeks of differential experience, the 3 groups of aged rats and young sedentary controls were tested for physical and cognitive function. Spatial discrimination learning and memory consolidation, tested on the water maze, were enhanced in environmentally-enriched compared with sedentary. A-EX exhibited improved and impaired performance on the cue and spatial task, respectively. Impaired spatial learning in A-EX was likely due to a bias in response selection associated with exercise training, as object recognition memory improved for A-EX rats. An examination of senescent hippocampal physiology revealed that enrichment and exercise reversed age-related changes in long-term depression (LTD) and long-term potentiation (LTP). Rats in the enrichment group exhibited an increase in cell excitability compared with the other 2 groups of aged animals. The results indicate that differential experience biased the selection of a spatial or a response strategy and factors common across the 2 conditions, such as increased hippocampal activity associated with locomotion, contribute to reversal of senescent synaptic plasticity.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) potently modulate the interactions of IGF-I and -II with the IGF-I receptor. Previous studies have shown that IGFBP2 gene expression is localized in astroglia, where it is anatomically and temporally coordinated with neuronal IGF-I expression during postnatal brain development. The present study shows that IGFBP5 gene expression is also highly abundant during brain development and also demonstrates significant spatiotemporal correlation with IGF-I, but exhibits a neuroanatomical distribution that is entirely distinct from IGFBP2. IGFBP5 and IGF-I mRNAs are synchronously coexpressed in principal neurons of sensory relay systems, including the olfactory bulb, medial and dorsal lateral geniculate bodies, and ventral tier, cochlear, lemniscal, and vestibular nuclei. They are also transiently coexpressed in principal neurons of the anterodorsal nucleus, but IGF-I mRNA disappears from this structure shortly after birth, while IGFBP5 mRNA remains highly abundant here in the adult. IGFBP5 and IGF-I gene expression demonstrate a temporally coordinated laminar association in the developing cerebellar cortex and hippocampal formation. IGF-I mRNA is concentrated in Purkinje cells, while IGFBP5 mRNA is localized in the external germinal zone in the developing cerebellar cortex. IGFBP5 mRNA is transiently expressed in the retrosplenial and cingulate cortex, subiculum, Ammon's horn, and amygdala, while IGF-I mRNA is contemporaneously localized in large interneurons distributed throughout the hippocampal formation. IGFBP5 mRNA is localized in the lateral ventricular germinal zone at birth and remains in the subventricular zone into maturity. It is also detected in forebrain white matter tracts and olfactory nerve from the second week after birth into maturity. Thus IGFBP5, in addition to IGFBP2, may be a significant determinant of IGF action in the brain. Colocalization in some sites and paralocalization with IGF-I in other sites suggests the potential for autocrine and paracrine interaction between the binding protein and IGF-I in different settings. Arguments are advanced suggesting a role for IGFBPs in the targeting of IGF action to specific cell addresses during brain development.

As neurons rely almost exclusively on glucose as an energy substrate, glucose transport is of critical importance to cerebral function. Two specific facilitative glucose transporters, GT1 and -3, predominate in brain, with the latter exclusively expressed by neurons, whereas GT1 is expressed by astrocytes and vascular elements. Little is known about the regulation of these transporters at the genetic level or the extent to which their expression may change in response to acute or chronic changes in metabolic demands. Thus, we employed in situ hybridization to evaluate changes in glucose transporter gene expression in the rat brain in response to ischemia induced by middle cerebral artery occlusion (MCAO). The most remarkable responses were demonstrated by GT1, which within an hour of ischemic insult demonstrated a global increase in gene expression throughout the forebrain. In the ensuing hours, GT1 expression further intensified and became lateralized to the lesioned hemisphere, with normalization of expression contralaterally. Increased GT1 mRNA levels were found in astroglia and microvessels and were also present in distinct neuronal populations, including the piriform cortex, dentate gyrus, and medial habenula, which normally do not express GT1 mRNA. By 24 h post-MCAO, glial cells of the ipsilateral cortex surrounding the infarct zone still demonstrated elevated GT1 mRNA levels, but expression had returned to baseline in neurons. Interestingly, it was not until GT1 expression had subsided (24 h post-MCAO), that there was a modest increase in neuronal GT3 gene expression in the affected hemisphere. GT2 and GT4 mRNAs were not detected in the rat brain under normal conditions or after ischemia. These data demonstrate that ischemia induces an immediate and sustained increase in brain GT1 gene expression in both glial cells and neurons. This augmentation of GT1 expression could represent a defensive strategy aimed at repletion of the brain's energy stores and stabilization of neuronal membrane potential.

The interactions of insulin-like growth factors (IGFs) with the type I IGF receptor are modulated by a family of high-affinity IGF binding proteins (IGFBPs). One of these, IGFBP2, demonstrates a striking spatiotemporal relationship with IGF-I during cerebellar and retinal development. IGF-I mRNA is transiently expressed in large projection neurons--cerebellar Purkinje and retinal ganglion cells--while IGFBP2 mRNA is selectively expressed by contiguous neuroglia--Bergmann glia in the cerebellum and Muller cells and astrocytes of the nerve fiber layer in the retina. IGF-I and IGFBP2 gene expression is not only neuroanatomically coordinated but also temporally synchronized, peaking together during the postnatal maturation of these structures. This pattern of IGF system expression suggests that IGFBP2 is closely related to IGF-I's action in the developing nervous system.

The tissue distribution and developmental patterns of insulin-like growth factor-II (IGF-II) have not been investigated in rat tissues, primarily because of the lack of an efficient extraction method for IGF-II and a sensitive RIA. IGF-II was extracted from rat tissues by formic acid, and the extract was heated at an acidic pH and treated with acetone. The removal of binding proteins was demonstrated by fast protein liquid chromatography size exclusion column and the elimination of a dilutional bias in the RIA. Using rat IGF-II as standard, we optimized a RIA for the quantification of IGF-II in rat tissues. In adult rats, IGF-II was found in all 15 tissues examined, with the highest concentration in the pituitary, followed by kidney, seminal vesicles, intestine, and serum. This distribution is not only different from that of IGF-I, but also differs from that reported for IGF-II mRNA and IGF-II receptors, suggesting that the rates of synthesis and/or metabolism of IGF-II are tissue dependent. Developmentally, IGF-II levels fell postnatally in most tissues, a pattern similar to that of IGF-II mRNA and IGF-II receptor. This developmental pattern supports the hypothesis that IGF-II is important in early growth and development. A relatively homogeneous distribution was observed in the adult rat brain, a distribution also different from that reported for IGF-II mRNA. In the pituitary, the highest concentration was found in the posterior pituitary, followed by the intermediate and anterior pituitary. In conclusion, IGF-II is found in many tissues of adult rats. This observation supports an autocrine and/or paracrine roles for IGF-II.

Analysis of insulin-like growth factors (IGF)-I, and -II, type II IGF receptor and IGF-binding protein-2 mRNA and peptide levels in normal and nephrectomized rat kidney. Immunocytochemistry, in situ hybridization, and radioimmunoassay were employed to examine the cellular distribution of mRNAs and proteins for IGF-I, II, IGF-II/M6P receptor, IGFBP2 as well as the levels of IGF-I and II in normal and unilaterally nephrectomized (Nx) adult rat kidneys. A similar distribution of immunoreactive IGF-I, and -II as well as IGF-II/M6P receptor was found in the principal cells of the cortical collecting duct and in all cells of the inner medullary collecting duct. In addition, immunostainable IGF-I and IGF-II/M6P receptor were noted in some inner medullary loops of Henle, while IGFBP2 was seen in the collecting ducts and loops of Henle of the inner medulla and the renal vasculature of all animals. By comparison, in situ hybridization revealed IGF-I mRNA only in the medullary thick ascending limbs while IGF-II mRNA was localized to the wall of the renal microvasculature in all kidneys. IGFBP2 mRNA was localized to the renal corpuscle and to inner medullary interstitial cells of all kidneys. These data suggest that renal IGF-I and IGFBP2 are synthesized at upstream sites along the nephron and then transported downstream for interaction with IGF receptors. Following nephrectomy, the renal levels of IGF-I peptide and mRNA were elevated at both 5 and 33 days post-nephrectomy, supporting a potential functional role for IGF-I in stimulating the structural and functional recovery in compensatory hypertrophy.