José A. Rafols

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury.

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.

Atrophy of medium spiny I striatal dendrites in advanced Parkinson's disease

Loss of dopaminergic neurons from the pars compacta of the substantia nigra is the pathological hallmark of Parkinsons disease (PD) and results in a partial deafferentation to the striatum. Since deafferentation is known to induce transynaptic atrophy of postsynaptic cells, we examined by Golgi impregnation the morphology of medium spiny I (MSI) striatal neurons, the principal target population for both nigrostriatal and corticostriatal fibers. Our quantitative data indicate that the dendritic arbor of MSI neurons in the putamen is significantly reduced in both length and number and MSI neurons are morphologically characterized by truncated dendrites with few dendritic spines and irregular, bulbous swellings. These data provide morphological evidence for the atrophy of striatal dendrites in PD and may explain, in part, the declining efficacy of chronic L-DOPA replacement therapy in advanced PD.

Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

The role of hypoxia-inducible factor-1α, aquaporin-4, and matrix metalloproteinase-9 in blood-brain barrier disruption and brain edema after traumatic brain injury

OBJECT The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α), aquaporin-4 (AQP-4), and matrix metalloproteinase-9 (MMP-9) in blood-brain barrier (BBB) permeability alterations and brain edema formation in a rodent traumatic brain injury (TBI) model. METHODS The brains of adult male Sprague-Dawley rats (400-425 g) were injured using the Marmarou closed-head force impact model. Anti-AQP-4 antibody, minocycline (an inhibitor of MMP-9), or 2-methoxyestradiol (2ME2, an inhibitor of HIF-1α), was administered intravenously 30 minutes after injury. The rats were killed 24 hours after injury and their brains were examined for protein expression, BBB permeability, and brain edema. Expression of HIF-1α, AQP-4, and MMP-9 as well as expression of the vascular basal lamina protein (laminin) and tight junction proteins (zona occludens-1 and occludin) was determined by Western blotting. Blood-brain barrier disruption was assessed by FITC-dextran extravasation, and brain edema was measured by the brain water content. RESULTS Significant (p < 0.05) edema and BBB extravasations were observed following TBI induction. Compared with sham-operated controls, the injured animals were found to have significantly (p < 0.05) enhanced expression of HIF-1α, AQP-4, and MMP-9, in addition to reduced amounts (p < 0.05) of laminin and tight junction proteins. Edema was significantly (p < 0.01) decreased after inhibition of AQP-4, MMP-9, or HIF-1α. While BBB permeability was significantly (p < 0.01) ameliorated after inhibition of either HIF-1α or MMP-9, it was not affected following inhibition of AQP-4. Inhibition of MMP reversed the loss of laminin (p < 0.01). Finally, while inhibition of HIF-1α significantly (p < 0.05) suppressed the expression of AQP-4 and MMP-9, such inhibition significantly (p < 0.05) increased the expression of laminin and tight junction proteins. CONCLUSIONS The data support the notion that HIF-1α plays a role in brain edema formation and BBB disruption via a molecular pathway cascade involving AQP-4 and MMP-9. Pharmacological blockade of this pathway in patients with TBI may provide a novel therapeutic strategy.

Cerebral angiogenesis and expression of angiogenic factors in aging rats after exercise.

The effect that exercise has on angiogenesis in the aging rat is unknown. We initiated this study with the intent to determine if exercise could induce angiogenesis in aging rats, as well as in adult rats reported previously. The markers we used to determine our endpoint were vascular endothelial growth factor (VEGF) and angiopoietin 1 and 2, as well as vascular density. Aged (22 month old) female Fisher 344 rats (n=16) were exercised on a treadmill 30 minutes each day for 3 weeks, or housed as non-exercised controls for the same duration. At the end of the exercise protocol, a significant (p<0.01) increase in the density of microvessels was found within the cerebral vasculature of the rats. Exercise was also associated with a significantly (p<0.01) increased mRNA expression of angiopoietin 1 and 2 in the aged cohort of rats. A mild but significant (p<0.01) increase in the four isoforms of VEGF mRNA (120, 144, 164, 188) were observed, with VEGF120 and VEGF144 being more markedly up-regulated than the other two. VEGF protein expression was also significantly (p<0.01) increased. This study demonstrates that angiogenesis can be induced in aging rats via exercise. The induced angiogenesis was associated with overexpression of angiogenic factors. These results support the hypothesis that an angiogenic response to chronic physical exercise is maintained with aging.

Effect of Brain Ischemia and Reperfusion on the Localization of Phosphorylated Eukaryotic Initiation Factor 2α

Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the α-subunit of eukaryotic initiation factor 2 (eIF-2α), we used an antibody specific to phosphorylated eIF-2α [eIF-2(αP)] to study the regional and cellular distribution of eIF-2(αP) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that ~1% of all eIF-2α is phosphorylated in controls, eIF-2(αP) is not reduced by up to 30 minutes of ischemia, and eIF-2(αP) is increased ~20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(αP) to astrocytes in normal brains, a massive increase in eIF-2(αP) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(αP) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(αP) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(αP) inhibits translation initiation, make a compelling case that eIF-2(αP) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.

Exercise-Induced Overexpression of Angiogenic Factors and Reduction of Ischemia / Reperfusion Injury in Stroke

The purpose of this study was to determine if exercise could induce expression of vascular endothelial growth factor (VEGF) and angiopoietin 1 and 2, in association with angiogenesis; and if angiogenic changes correlated with reduced brain injury in stroke. Adult male Sprague Dawley rats (3 month old, n=44) were exercised on a treadmill 30 minutes each day for 1, 3 or 6 weeks, or housed as non-exercised controls for 3 weeks. Some 3 week-exercised rats were then housed for an additional 3 weeks. Exercise significantly (p<0.01) increased mRNA (determined by real-time reverse transcriptase-polymerase chain reaction) expression of angiopoietin 1 and 2 as early as 1 week, with further increases occurring at 3 weeks. A mild increase after 1 week and a robust increase after 3 weeks of exercise in four isoforms (120, 144, 164, 188) of VEGF mRNA levels were significantly (p<0.01) observed, with VEGF(144) being more markedly up-regulated. Overexpression of the mRNAs decreased upon withdrawal of exercise. A significant increase (p<0.01) in the density of microvessels (determined by laminin-immunocytochemistry) was found at 3 weeks of exercise and this continued after exercise was withdrawn. In exercising rats subjected to 2-h MCA occlusion followed by 48-h reperfusion, neurological deficits and infarct volume were significantly reduced. Neuroprotection continued after 3 weeks of rest. This study indicates that pre-ischemic exercise reduces brain injury in stroke. The reduced damage is associated with angiogenesis, possibly induced by angiogenic factors following exercise. Physical exercise up-regulates mRNA levels of the angiopoietin family and VEGF.

Increased astrocyte proliferation in rats after running exercise

The aim in this study was to investigate whether physical exercise could induce astroglial proliferation in the frontoparietal cortex and dorsolateral striatum where extensive angiogenesis had been found after exercise in previous studies. Adult male Sprague Dawley rats (n=48) were used in four experimental groups. Animals were exercised 30 min each day on a treadmill on which repetitive locomotor movement was required, for 0 (n=12), 3 (n=12) or 6 (n=12) weeks, as well as 3-week exercise plus 3-week rest (n=12). Brain tissues of the exercised and non-exercised rats were processed for glial fibrillary acidic protein (GFAP) immunocytochemistry (n=6 x 4) and Western blotting (n=6 x 4) to evaluate regional astrocyte proliferation in the frontoparietal cortex and dorsolateral striatum. By using GFAP immunocytochemistry and stereological methods, we compared the density of astrocytes in the animals with or without exercise. In comparison to non-exercised animals, a significant (p<0.01) increase in the number of astrocytes was observed in both cortex and striatum of rats exercised for 3 or 6 weeks. Our data also indicated that astrocytic density continued to increase up to 6 weeks either with an additional 3 weeks of exercise (p<0.01) or 3 weeks of rest (p<0.01). In addition, Western blotting analysis showed an obvious increase in GFAP protein from cortex and striatum of exercised animals. Astrocytosis after exercise, coupled with angiogenesis, is thought to provide strength to the neurovascular unit (a construct consisting of microvascular endothelium, astroglia, neurons and the extracellular matrix). Strengthening of this unit by exercise may protect blood-brain-barrier function following brain injury, such as that occurring after stroke.

The ultrastructure and synaptic architecture of phrenic motor neurons in the spinal cord of the adult rat

SummaryAlthough light microscopic studies have analysed phrenic motor neurons in several different species, there has never been an ultrastructural investigation of identified phrenic motor neurons. In addition, electrophysiological studies have raised questions relating to the function of phrenic motor neurons which may be answered only by direct electron microscopic investigation. Thus, the present study was carried out to provide a detailed ultrastructural analysis of identified phrenic motor neurons. Phrenic motor neurons in the spinal cord of the rat were labelled by retrogradely transported horseradish peroxidase (HRP) after transecting the phrenic nerve in the neck and applying the enzyme directly to the central stump of the transected nerve. The results showed that the general ultrastructural characteristics of phrenic motor neurons were similar to those previously reported for other spinal motor neurons. However, phrenic primary dendrites appeared to be isolated from all other dendritic profiles in the neuropil. Primary dendrites were not fasciculated. Fasciculation occurred only among the more distal secondary and tertiary phrenic dendritic branches. Direct dendrodendritic or dendrosomatic apposition was rarely seen; gap junctions between directly apposing phrenic neuronal membranes were not observed. The membranes of adjacent phrenic neuronal profiles were most frequently separated by intervening sheaths of astroglial processes. Myelinated phrenic axons and a phrenic axon collateral were identified. The initial portion of the phrenic axon collateral was cone-shaped, lacked myelin, and thus resembled a miniature axon hillock. In one instance, a large accumulation of polyribosomes was observed within the hillock-like structure of a phrenic axon collateral. Eight morphological types of synaptic boutons, M, P, NFs, S, NFf, F, G and C were classified according to criteria used by previous investigators. Most of these endings (M, NFs, NFf, S and F) made synaptic contact with profiles of labelled phrenic somata and dendrites. F, NFf, and S boutons also terminated on phrenic axon hillocks. C and G boutons contacted exclusively phrenic somata and small calibre dendrites, respectively. P boutons established axo-axonic synaptic contacts with the M and NFs bouton. The morphological findings of the present study provide new data that may be related to phrenic synchronized output and presynaptic inhibition of primary afferents terminating on phrenic motor neurons.

High fat feeding is associated with increased blood pressure, sympathetic nerve activity and hypothalamic mu opioid receptors

Obesity and high fat diets are associated with an increased prevalence of diabetes, cardiovascular disease, and hypertension. However, the mechanism(s) linking obesity and high fat diet to these metabolic and cardiovascular disorders are not fully elucidated. Leptin stimulates the formation of pro-opiomelanocortin and its products. The stimulation of the central nervous system (CNS) opioids and their receptors is associated with an increase in cardiovascular dynamics. In this study we hypothesized that obesity changed the CNS opioids and their receptors that could play a role in altered cardiovascular and autonomic nervous regulation in obesity. Male Wistar rats were fed either a high fat (HF) or regular chow (control) diet. After 12 weeks, rats were anesthetized and instrumented to record mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). A blood sample was collected and plasma glucose, insulin, leptin, beta-endorphins were measured. The brains were subsequently processed for immunohistochemistry and in situ hybridization. The HF rats were larger and had a greater percentage of body fat. Leptin and insulin levels were also higher in the HF animals. Basal MAP and RSNA were significantly higher in HF rats. Additionally, immunohistochemistry and in situ hybridization demonstrated that HF rats had increased hypothalamus mu opioid receptors compared to controls. These studies suggest that HF feeding is associated with increased body fat, plasma leptin, insulin, and hypothalamic mu opioid receptors. The increased mu opioid receptors may contribute to the higher MAP and RSNA observed in HF animals.