Rehabilitation Augments Hematoma Clearance and Attenuates Oxidative Injury and Ion Dyshomeostasis After Brain Hemorrhage.

BACKGROUND AND PURPOSE: We assessed the elemental and biochemical effects of rehabilitation after intracerebral hemorrhage, with emphasis on iron-mediated oxidative stress, using a novel multimodal biospectroscopic imaging approach. METHODS: Collagenase-induced striatal hemorrhage was produced in rats that were randomized to enriched rehabilitation or control intervention starting on day 7. Animals were euthanized on day 14 or 21, a period of ongoing cell death. We used biospectroscopic imaging techniques to precisely determine elemental and molecular changes on day 14. Hemoglobin content was assessed with resonance Raman spectroscopy. X-ray fluorescence imaging mapped iron, chlorine, potassium, calcium, and zinc. Protein aggregation, a marker of oxidative stress, and the distribution of other macromolecules were assessed with Fourier transform infrared imaging. A second study estimated hematoma volume with a spectrophotometric assay at 21 days. RESULTS: In the first experiment, rehabilitation reduced hematoma hemoglobin content (P=0.004) and the amount of peri-hematoma iron (P<0.001). Oxidative damage was highly localized at the hematoma/peri-hematoma border and was decreased by rehabilitation (P=0.004). Lipid content in the peri-hematoma zone was increased by rehabilitation (P=0.016). Rehabilitation reduced the size of calcium deposits (P=0.040) and attenuated persistent dyshomeostasis of Cl- (P<0.001) but not K+ (P=0.060). The second study confirmed that rehabilitation decreased hematoma volume (P=0.024). CONCLUSIONS: Rehabilitation accelerated clearance of toxic blood components and decreased chronic oxidative stress. As well, rehabilitation attenuated persistent ion dyshomeostasis. These novel effects may underlie rehabilitation-induced neuroprotection and improved recovery of function. Pharmacotherapies targeting these mechanisms may further improve outcome.

A novel multi-modal platform to image molecular and elemental alterations in ischemic stroke.

Stroke is a major global health problem, with the prevalence and economic burden predicted to increase due to aging populations in western society. Following stroke, numerous biochemical alterations occur and damage can spread to nearby tissue. This zone of "at risk" tissue is termed the peri-infarct zone (PIZ). As the PIZ contains tissue not initially damaged by the stroke, it is considered by many as salvageable tissue. For this reason, much research effort has been undertaken to improve the identification of the PIZ and to elucidate the biochemical mechanisms that drive tissue damage in the PIZ in the hope of identify new therapeutic targets. Despite this effort, few therapies have evolved, attributed in part, to an incomplete understanding of the biochemical mechanisms driving tissue damage in the PIZ. Magnetic resonance imaging (MRI) has long been the gold standard to study alterations in gross brain structure, and is frequently used to study the PIZ following stroke. Unfortunately, MRI does not have sufficient spatial resolution to study individual cells within the brain, and reveals little information on the biochemical mechanisms driving tissue damage. MRI results may be complemented with histology or immuno-histochemistry to provide information at the cellular or sub-cellular level, but are limited to studying biochemical markers that can be successfully "tagged" with a stain or antigen. However, many important biochemical markers cannot be studied with traditional MRI or histology/histochemical methods. Therefore, we have developed and applied a multi-modal imaging platform to reveal elemental and molecular alterations that could not previously be imaged by other traditional methods. Our imaging platform incorporates a suite of spectroscopic imaging techniques; Fourier transform infrared imaging, Raman spectroscopic imaging, Coherent anti-stoke Raman spectroscopic imaging and X-ray fluorescence imaging. This approach does not preclude the use of traditional imaging techniques, and rather it should be use to complement traditional methods such as MRI or histology and immunohistochemistry, to gain a greater insight into disease mechanisms. We demonstrate the potential of this approach by characterizing biochemical alterations within the PIZ 24h after the induction of photothrombotic stroke in mice. Substantial molecular and elemental alterations were identified in the PIZ 24h after stroke that are consistent with tissue swelling and edema, but not oxidative stress. This reveals important mechanistic information, that could not previously be obtained, which should be considered in future studies aimed at developing therapeutic intervention from this model.

Design of a mouse restraint for synchrotron-based computed tomography imaging.

High-resolution computed tomography (CT) imaging of a live animal within a lead-lined synchrotron light hutch presents several unique challenges. In order to confirm that the animal is under a stable plane of anaesthesia, several physiological parameters (e.g. heart rate, arterial oxygen saturation, core body temperature and respiratory rate) must be remotely monitored from outside the imaging hutch. In addition, to properly scan the thoracic region using CT, the animal needs to be held in a vertical position perpendicular to the fixed angle of the X-ray beam and free to rotate 180°-360°. A new X-ray transparent mouse restraint designed and fabricated using computer-aided design software and three-dimensional rapid prototype printing has been successfully tested at the Biomedical Imaging and Therapy bending-magnet (BMIT-BM) beamline at the Canadian Light Source.

Measuring iron in the brain using quantitative susceptibility mapping and X-ray fluorescence imaging.

Measuring iron content in the brain has important implications for a number of neurodegenerative diseases. Quantitative susceptibility mapping (QSM), derived from magnetic resonance images, has been used to measure total iron content in vivo and in post mortem brain. In this paper, we show how magnetic susceptibility from QSM correlates with total iron content measured by X-ray fluorescence (XRF) imaging and by inductively coupled plasma mass spectrometry (ICPMS). The relationship between susceptibility and ferritin iron was estimated at 1.10±0.08 ppb susceptibility per µg iron/g wet tissue, similar to that of iron in fixed (frozen/thawed) cadaveric brain and previously published data from unfixed brains. We conclude that magnetic susceptibility can provide a direct and reliable quantitative measurement of iron content and that it can be used clinically at least in regions with high iron content.

Susceptibility weighted MRI pinpoints spontaneous intracerebral hemorrhage in stroke-prone spontaneously hypertensive rats.

PURPOSE: To find magnetic resonance imaging (MRI) precursors of spontaneous intracerebral hemorrhage in stroke-prone spontaneously hypertensive rats (SHRSP). METHOD: SHRSP rats were used with both a low/high salt (n = 18 or 11) Japanese diet and salty drinking water to generate spontaneous intracerebral hemorrhage (ICH). Various MRI sequences, and in particular, susceptibility weighted imaging (SWI), were used and combined with a gadolinium (Gd) contrast agent or oxygen gas to identify the rupture of the blood brain barrier (BBB) and the temporal ICH. RESULTS: Most rats developed hypertensive ICH stroke in the high salt group during the 10-13 week period compared to only one third of rats in the low salt group during the 14-18 week period. The location of stroke for both the low/high-salt groups was highest in the striatum (58%/43%), followed by the cortex (21%/30%). The edematous enhancement on T2 weighted (T2W) imaging or Gd based T1 weighted (Gd-T1W) imaging due to the ruptured BBB preceded the striatal hemorrhages seen on SWI. The most recent bleeds were observed on temporal SWI or on oxygen-enhanced SWI. The mode of the volume of bleeds was 0.4 mm3. A positive correlation between susceptibility x volume and R2* x volume of the bleeds was observed. CONCLUSIONS: SHRSP rats with the high salt diet effectively generated a hypertensive hemorrhagic stroke which could be monitored by various MRI sequences. The venous dilation on SWI may precede any abnormality on T2W or Gd-T1W imaging. The edematous enhancement on T2W or Gd-T1W indicated a BBB breakdown that may precede striatal ICH by several days. This suggests the need for immediate treatment to improve outcome if this finding is observed. The use of oxygen with SWI was able to help differentiate old bleeds from very recent bleeds.

Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age.

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a 'sub-proteome', such as the 'metalloproteome', defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1-1) isoform in 15% of the population.

Brain iron detected by SWI high pass filtered phase calibrated with synchrotron X-ray fluorescence.

PURPOSE: To test the ability of susceptibility weighted images (SWI) and high pass filtered phase images to localize and quantify brain iron. MATERIALS AND METHODS: Magnetic resonance (MR) images of human cadaver brain hemispheres were collected using a gradient echo based SWI sequence at 1.5T. For X-ray fluorescence (XRF) mapping, each brain was cut to obtain slices that reasonably matched the MR images and iron was mapped at the iron K-edge at 50 or 100 microm resolution. Iron was quantified using XRF calibration foils. Phase and iron XRF were averaged within anatomic regions of one slice, chosen for its range of iron concentrations and nearly perfect anatomic correspondence. X-ray absorption spectroscopy (XAS) was used to determine if the chemical form of iron was different in regions with poorer correspondence between iron and phase. RESULTS: Iron XRF maps, SWI, and high pass filtered phase data in nine brain slices from five subjects were visually very similar, particularly in high iron regions. The chemical form of iron could not explain poor matches. The correlation between the concentration of iron and phase in the cadaver brain was estimated as c(Fe) [microg/g tissue] = 850Deltavarpi + 110. CONCLUSION: The phase shift Deltavarpi was found to vary linearly with iron concentration with the best correspondence found in regions with high iron content.

PBT2 acts through a different mechanism of action than other 8-hydroxyquinolines: an X-ray fluorescence imaging study.

8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.

Synchrotron X-ray fluorescence reveals abnormal metal distributions in brain and spinal cord in spinocerebellar ataxia: a case report.

For the first time, synchrotron rapid-scanning X-ray fluorescence (RS-XRF) was used to simultaneously localize and quantify iron, copper, and zinc in spinal cord and brain in a case of spinocerebellar ataxia (SCA). In the normal medulla, a previously undescribed copper enrichment was seen associated with spinocerebellar fibers and amiculum olivae. This region was virtually devoid of all metals in the SCA case. Regions with neuronal loss and gliosis in the cerebellar cortex, inferior olivary, and dentate nuclei and areas showing loss of myelinated fibers were also low in all metals in SCA compared to control. In contrast, the ventral columns of the spinal cord that exhibited only moderate myelin pallor had increased metal levels. Iron and zinc were also elevated in the globus pallidus pars externa in SCA relative to control. We hypothesize that metals increase as part of the initial neurodegenerative process, but once degeneration is advanced, the metal levels drop. This implies a role for multiple metals in SCA neurodegeneration, but further study is required to establish a causative role. We suggest that if these findings are generally true of at least some cases of SCA, not only iron but also copper and zinc should be considered as possible therapeutic targets.

Iron, copper, and zinc distribution of the cerebellum.

Synchrotron rapid-scanning X-ray fluorescence (RS-XRF) is employed for the first time to simultaneously map iron, copper, and zinc in the normal cerebellum. The cerebellum is a major repository of metals that are essential to normal function. Therefore, mapping the normal metal distribution is an important first step towards understanding how multiple metals may induce oxidative damage, protein aggregation, and neurotoxicity leading to cerebellar degeneration in a wide range of diseases. We found that cerebellar white and grey matter could be sharply defined based upon the unique metal content of each region. The dentate nucleus was particularly metal-rich with copper localized to the periphery and iron and zinc abundant centrally. We discuss how RS-XRF metal mapping in the normal brain may yield important clues to the mechanisms of degeneration in the dentate nucleus.

Mapping metals in Parkinson's and normal brain using rapid-scanning x-ray fluorescence.

Rapid-scanning x-ray fluorescence (RS-XRF) is a synchrotron technology that maps multiple metals in tissues by employing unique hardware and software to increase scanning speed. RS-XRF was validated by mapping and quantifying iron, zinc and copper in brain slices from Parkinson's disease (PD) and unaffected subjects. Regions and structures in the brain were readily identified by their metal complement and each metal had a unique distribution. Many zinc-rich brain regions were low in iron and vice versa. The location and amount of iron in brain regions known to be affected in PD agreed with analyses using other methods. Sample preparation is simple and standard formalin-fixed autopsy slices are suitable. RS-XRF can simultaneously and non-destructively map and quantify multiple metals and holds great promise to reveal metal pathologies associated with PD and other neurodegenerative diseases as well as diseases of metal metabolism.

Prolonged Blood-Brain Barrier Injury Occurs After Experimental Intracerebral Hemorrhage and Is Not Acutely Associated with Additional Bleeding.

Intracerebral hemorrhage (ICH) causes blood-brain barrier (BBB) damage along with altered element levels in the brain. BBB permeability was quantified at 3, 7, and 14 days with Evans Blue dye after collagenase-induced ICH in rat. At peak permeability (day 3), a gadolinium (Gd)-based contrast agent was injected to further characterize BBB disruption, and X-ray fluorescence imaging (XFI) was used to map Gd, Fe, Cl, and other elements. XFI revealed that Ca, Cl, Gd, and Fe concentrations were significantly elevated, whereas K was significantly decreased. Therefore, using Gd-XFI, we co-determined BBB dysfunction with alterations in the metallome, including those that contribute to cell death and functional outcome. Warfarin was administered 3 days post-ICH to investigate whether additional or new bleeding occurs during peak BBB dysfunction, and hematoma volume was assessed on day 4. Warfarin administration prolonged bleeding time after a peripheral cut-induced bleed, but warfarin did not worsen hematoma volume. Accordingly, extensive BBB leakage occurred after ICH, but did not appear to affect total hematoma size.

Protein-Energy Malnutrition Exacerbates Stroke-Induced Forelimb Abnormalities and Dampens Neuroinflammation.

Protein-energy malnutrition (PEM) pre-existing at stroke onset is believed to worsen functional outcome, yet the underlying mechanisms are not fully understood. Since brain inflammation is an important modulator of neurological recovery after stroke, we explored the impact of PEM on neuroinflammation in the acute period in relation to stroke-initiated sensori-motor abnormalities. Adult rats were fed a low-protein (LP) or normal protein (NP) diet for 28 days before inducing photothrombotic stroke (St) in the forelimb region of the motor cortex or sham surgery; the diets continued for 3 days after the stroke. Protein-energy status was assessed by a combination of body weight, food intake, serum acute phase proteins and corticosterone, and liver lipid content. Deficits in motor function were evaluated in the horizontal ladder walking and cylinder tasks at 3 days after stroke. The glial response and brain elemental signature were investigated by immunohistochemistry and micro-X-ray fluorescence imaging, respectively. The LP-fed rats reduced food intake, resulting in PEM. Pre-existing PEM augmented stroke-induced abnormalities in forelimb placement accuracy on the ladder; LP-St rats made more errors (29 ± 8%) than the NP-St rats (15 ± 3%; P < 0.05). This was accompanied by attenuated astrogliosis in the peri-infarct area by 18% and reduced microglia activation by up to 41 and 21% in the peri-infarct area and the infarct rim, respectively (P < 0.05). The LP diet altered the cortical Zn, Ca, and Cl signatures (P < 0.05). Our data suggest that proactive treatment of pre-existing PEM could be essential for optimal post-stroke recovery.

The chemical form of mitochondrial iron in Friedreich's ataxia.

Friedreich's ataxia (FRDA) results from cellular damage caused by a deficiency in the mitochondrial matrix protein frataxin. To address the effect of frataxin deficiency on mitochondrial iron chemistry, the heavy mitochondrial fraction (HMF) was isolated from primary fibroblasts from FRDA affected and unaffected individuals. X-ray absorption spectroscopy was used to characterize the chemical form of iron. Near K-edge spectra were fitted with a series of model iron compounds to determine the proportion of each iron species. Most of the iron in both affected and unaffected fibroblasts was ferrihydrite. The iron K-edge from unaffected HMFs were best fitted with poorly organized ferrihydrite modeled by frataxin whereas HMFs from affected cells were best fitted with highly organized ferrihydrite modeled by ferritin. Both had several minor iron species but these did not differ consistently with disease. Since the iron K-edge spectra of ferritin and frataxin are very similar, we present additional evidence for the presence of ferritin-bound iron in HMF. The predominant ferritin subunit in HMFs from affected cells resembled mitochondrial ferritin (MtFt) in size and antigenicity. Western blotting of native gels showed that HMF from affected cells had 3-fold more holoferritin containing stainable iron. We conclude that most of the iron in fibroblast HMF from both affected and unaffected cells is ferrihydrite but only FRDA affected cells mineralize significant iron in mitochondrial ferritin.

Examining potential side effects of therapeutic hypothermia in experimental intracerebral hemorrhage.

Studies treating intracerebral hemorrhage (ICH) with therapeutic hypothermia (TH) have shown inconsistent benefits. We hypothesized that TH's anti-inflammatory effects may be responsible as inflammatory cells are essential for removing degrading erythrocytes. Here, we subjected rats to a collagenase-induced striatal ICH followed by whole-body TH (∼33℃ for 11-72 h) or normothermia. We used X-ray fluorescence imaging to spatially quantify total and peri-hematoma iron three days post-injury. At three and seven days, we measured non-heme iron levels. Finally, hematoma volume was quantified on one, three, and seven days. In the injured hemisphere, total iron levels were elevated ( p < 0.001) with iron increasing in the peri-hematoma region ( p = 0.007). Non-heme iron increased from three to seven days (p < 0.001). TH had no effect on any measure of iron ( p ≥ 0.479). At one and three days, TH did not affect hematoma volume ( p ≥ 0.264); however, at seven days there was a four-fold increase in hematoma volume in 40% of treated animals ( p = 0.032). Thus, even when TH does not interfere with initial increases in total and non-heme iron or its containment, TH can cause re-bleeding post-treatment. This serious complication could partly account for the intermittent protection previously observed. This also raises serious concerns for clinical usage of TH for ICH.

A new method to image heme-Fe, total Fe, and aggregated protein levels after intracerebral hemorrhage.

An intracerebral hemorrhage (ICH) is a devastating stroke that results in high mortality and significant disability in survivors. Unfortunately, the underlying mechanisms of this injury are not yet fully understood. After the primary (mechanical) trauma, secondary degenerative events contribute to ongoing cell death in the peri-hematoma region. Oxidative stress is thought to be a key reason for this delayed injury, which is likely due to free-Fe-catalyzed free radical reactions. Unfortunately, this is difficult to prove with conventional biochemical assays that fail to differentiate between alterations that occur within the hematoma and peri-hematoma zone. This is a critical limitation, as the hematoma contains tissue severely damaged by the initial hemorrhage and is unsalvageable, whereas the peri-hematoma region is less damaged but at risk from secondary degenerative events. Such events include oxidative stress mediated by free Fe presumed to originate from hemoglobin breakdown. Therefore, minimizing the damage caused by oxidative stress following hemoglobin breakdown and Fe release is a major therapeutic target. However, the extent to which free Fe contributes to the pathogenesis of ICH remains unknown. This investigation used a novel imaging approach that employed resonance Raman spectroscopic mapping of hemoglobin, X-ray fluorescence microscopic mapping of total Fe, and Fourier transform infrared spectroscopic imaging of aggregated protein following ICH in rats. This multimodal spectroscopic approach was used to accurately define the hematoma/peri-hematoma boundary and quantify the Fe concentration and the relative aggregated protein content, as a marker of oxidative stress, within each region. The results revealed total Fe is substantially increased in the hematoma (0.90 µg cm(-2)), and a subtle but significant increase in Fe that is not in the chemical form of hemoglobin is present within the peri-hematoma zone (0.32 µg cm(-2)) within 1 day of ICH, relative to sham animals (0.22 µg cm(-2)). Levels of aggregated protein were significantly increased within both the hematoma (integrated band area 0.10 AU) and peri-hematoma zone (integrated band area 0.10 AU) relative to sham animals (integrated band area 0.056 AU), but no significant difference in aggregated protein content was observed between the hematoma and peri-hematoma zone. This result suggests that the chemical form of Fe and its ability to generate free radicals is likely to be a more critical predictor of tissue damage than the total Fe content of the tissue. Furthermore, this article describes a novel approach to colocalize nonheme Fe and aggregated protein in the peri-hematoma zone following ICH, a significant methodological advancement for the field.

In situ biospectroscopic investigation of rapid ischemic and postmortem induced biochemical alterations in the rat brain.

Rapid advances in imaging technologies have pushed novel spectroscopic modalities such as Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (XAS) at the sulfur K-edge to the forefront of direct in situ investigation of brain biochemistry. However, few studies have examined the extent to which sample preparation artifacts confound results. Previous investigations using traditional analyses, such as tissue dissection, homogenization, and biochemical assay, conducted extensive research to identify biochemical alterations that occur ex vivo during sample preparation. In particular, altered metabolism and oxidative stress may be caused by animal death. These processes were a concern for studies using biochemical assays, and protocols were developed to minimize their occurrence. In this investigation, a similar approach was taken to identify the biochemical alterations that are detectable by two in situ spectroscopic methods (FTIR, XAS) that occur as a consequence of ischemic conditions created during humane animal killing. FTIR and XAS are well suited to study markers of altered metabolism such as lactate and creatine (FTIR) and markers of oxidative stress such as aggregated proteins (FTIR) and altered thiol redox (XAS). The results are in accordance with previous investigations using biochemical assays and demonstrate that the time between animal death and tissue dissection results in ischemic conditions that alter brain metabolism and initiate oxidative stress. Therefore, future in situ biospectroscopic investigations utilizing FTIR and XAS must take into consideration that brain tissue dissected from a healthy animal does not truly reflect the in vivo condition, but rather reflects a state of mild ischemia. If studies require the levels of metabolites (lactate, creatine) and markers of oxidative stress (thiol redox) to be preserved as close as possible to the in vivo condition, then rapid freezing of brain tissue via decapitation into liquid nitrogen, followed by chiseling the brain out at dry ice temperatures is required.

Novel bio-spectroscopic imaging reveals disturbed protein homeostasis and thiol redox with protein aggregation prior to hippocampal CA1 pyramidal neuron death induced by global brain ischemia in the rat.

Global brain ischemia resulting from cardiac arrest and cardiac surgery can lead to permanent brain damage and mental impairment. A clinical hallmark of global brain ischemia is delayed neurodegeneration, particularly within the CA1 subsector of the hippocampus. Unfortunately, the biochemical mechanisms have not been fully elucidated, hindering optimization of current therapies (i.e., therapeutic hypothermia) or development of new therapies. A major limitation to elucidating the mechanisms that contribute to neurodegeneration and understanding how these are influenced by potential therapies is the inability to relate biochemical markers to alterations in the morphology of individual neurons. Although immunocytochemistry allows imaging of numerous biochemical markers at the sub-cellular level, it is not a direct chemical imaging technique and requires successful "tagging" of the desired analyte. Consequently, important biochemical parameters, particularly those that manifest from oxidative damage to biological molecules, such as aggregated protein levels, have been notoriously difficult to image at the cellular or sub-cellular level. It has been hypothesized that reactive oxygen species (ROS) generated during ischemia and reperfusion facilitate protein aggregation, impairing neuronal protein homeostasis (i.e., decreasing protein synthesis) that in turn promotes neurodegeneration. Despite indirect evidence for this theory, direct measurements of morphology and ROS induced biochemical damage, such as increased protein aggregates and decreased protein synthesis, within the same neuron is lacking, due to the unavailability of a suitable imaging method. Our experimental approach has incorporated routine histology with novel wide-field synchrotron radiation Fourier transform infrared imaging (FTIRI) of the same neurons, ex vivo within brain tissue sections. The results demonstrate for the first time that increased protein aggregation and decreased levels of total protein occur in the same CA1 pyramidal neurons 1 day after global ischemia. Further, analysis of serial tissue sections using X-ray absorption spectroscopy at the sulfur K-edge has revealed that CA1 pyramidal neurons have increased disulfide levels, a direct indicator of oxidative stress, at this time point. These changes at 1 day after ischemia precede a massive increase in aggregated protein and disulfide levels concomitant with loss of neuron integrity 2 days after ischemia. Therefore, this study has provided direct support for a correlative mechanistic link in both spatial and temporal domains between oxidative stress, protein aggregation and altered protein homeostasis prior to irreparable neuron damage following global ischemia.

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation.

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60-100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90-156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.

The solution structure of the copper clioquinol complex.

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) recently has shown promising results in the treatment of Alzheimer's disease and in cancer therapy, both of which also are thought to be due to clioquinol's ability as a lipophilic copper chelator. Previously, clioquinol was used as an anti-fungal and anti-protozoal drug that was responsible for an epidemic of subacute myelo-optic neuropathy (SMON) in Japan during the 1960s, probably a myeloneuropathy arising from a clioquinol-induced copper deficiency. Previous X-ray absorption spectroscopy of solutions of copper chelates of clioquinol suggested unusual coordination chemistry. Here we use a combination of electron paramagnetic, UV-visible and X-ray absorption spectroscopies to provide clarification of the chelation chemistry between clioquinol and copper. We find that the solution structures for the copper complexes formed with stoichiometric and excess clioquinol are conventional 8-hydroxyquinolate chelates. Thus, the promise of clioquinol in new treatments for Alzheimer's disease and in cancer therapy is not likely to be due to any novel chelation chemistry, but rather due to other factors including the high lipophilicity of the free ligand and chelate complexes.