An in vivo model of anti-inflammatory activity of subdural dexamethasone following the spinal cord injury

Current therapies to limit the neural tissue destruction following the spinal cord injury are not effective. Our recent studies indicate that the injury to the white matter of the spinal cord results in a severe inflammatory response where macrophages phagocytize damaged myelin and the fluid-filled cavity of injury extends in size with concurrent and irreversible destruction of the surrounding neural tissue over several months. We previously established that a high dose of 4mg/rat of dexamethasone administered for 1 week via subdural infusion remarkably lowers the numbers of infiltrating macrophages leaving large amounts of un-phagocytized myelin debris and therefore inhibits the severity of inflammation and related tissue destruction. But this dose was potently toxic to the rats. In the present study the lower doses of dexamethasone, 0.125–2.0mg, were administered via the subdural infusion for 2 weeks after an epidural balloon crush of the mid-thoracic spinal cord. The spinal cord cross-sections were analyzed histologically. Levels of dexamethasone used in the current study had no systemic toxic effect and limited phagocytosis of myelin debris by macrophages in the lesion cavity. The subdural infusion with 0.125–2.0mg dexamethasone over 2 week period did not eliminate the inflammatory process indicating the need for a longer period of infusion to do so. However, this treatment has probably lead to inhibition of the tissue destruction by the severe, prolonged inflammatory process

High Resolution Quantitative MRI in a Non-Surgical Model of Spinal Cord Injury

Magnetic resonance imaging (MRI) is very sensitive to the presence of damage resulting from injury or disease, but often lacks specificity. Quantitative MRI can significantly increase the specificity in the presence of pathology but must be validated, often using an animal model, for each type of injury or disease. In the case of spinal cord injury (SCI) most models are difficult to image, either due to the location of the injury, or as a result of damage to surrounding tissues resulting from invasive surgical procedures. This thesis describes a non-surgical model of rat SCI which uses MR guided focused ultrasound and microbubbles to create an injury the cervical spinal cord which is optimal for performing quantitative MRI, and compares it with other models of SCI using MRI, histology, and immunohistochemistry. It also describes the difficulties encountered when implementing the quantitative T2 (qT2) MR sequence at the very high resolution required to image the rat spinal cord, the limitations on the qT2 sequence due to the presence of diffusion, and how the effects of diffusion were minimized. Using the new SCI model and qT2 sequence, qT2 and diffusion data were acquired at 24 hours, 1 week, and 2 weeks following SCI, and the quantitative MRI parameters were correlated with histology. The increased specificity gained using quantitative MRI will increase the information available at each timepoint, reducing both the variability and cost of longitudinal studies aimed at developing treatments for SCI.Ph.D

Intraindividual variability of striatal 1H-MRS brain metabolite measurements at 3 T

Purpose To measure possible positional and diurnal physiological effects on brain metabolites in single-voxel proton magnetic resonance spectroscopy (1H-MRS) measurements of the right and left striatum. Methods 1H-MRS measurements were performed in 10 healthy adult volunteers using a short echo PRESS sequence (TE=30 ms, TR=3000 ms). Each individual was scanned during both morning and afternoon hours. Regions of interest were right and left striatum. To control for systematic drift in scanner performance, 1H-MRS measurements of a standard phantom solution were also acquired. Statistical analysis was performed using a repeated measures analysis of variance that included three within-subject factors: metabolite (N-acetyl-aspartate [NAA] or creatine [Cr]), laterality (left or right caudate) and time (morning or afternoon). Results A significant interaction (P<.016) between time of day and metabolite levels was observed. Further exploration of this finding revealed a significant difference between morning and afternoon levels of NAA (P<.044) but not Cr. In addition, no significant morning-to-afternoon differences were observed for the 1H-MRS phantom measurements. Conclusions Systematic variation due to scanner performance does not account for the changes observed in repeated measurements of striatal NAA levels. This difference may be accounted for by either repositioning effects or circadian physiological effects. Further studies are required to learn whether time of day standardization of 1H-MRS acquisitions may contribute to improved reproducibility of measurements

Prolonged Subdural Infusion of Kynurenic Acid Is Associated with Dose-Dependent Myelin Damage in the Rat Spinal Cord.

Kynurenic acid (KYNA) is the end stage metabolite of tryptophan produced mainly by astrocytes in the central nervous system (CNS). It has neuroprotective activities but can be elevated in the neuropsychiatric disorders. Toxic effects of KYNA in the CNS are unknown. The aim of this study was to assess the effect of the subdural KYNA infusion on the spinal cord in adult rats.A total of 42 healthy adult rats were randomly assigned into six groups and were infused for 7 days with PBS (control) or 0.0002 pmol/min, 0.01 nmol/min, 0.1 nmol/min, 1 nmol/min, and 10 nmol/min of KYNA per 7 days. The effect of KYNA on spinal cord was determined using histological and electron microscopy examination. Myelin oligodendrocyte glycoprotein (MOG) was measured in the blood serum to assess a degree of myelin damage.In all rats continuous long-lasting subdural KYNA infusion was associated with myelin damage and myelin loss that was increasingly widespread in a dose-depended fashion in peripheral, sub-pial areas. Damage to myelin sheaths was uniquely related to the separation of lamellae at the intraperiod line. The damaged myelin sheaths and areas with complete loss of myelin were associated with limited loss of scattered axons while vast majority of axons in affected areas were morphologically intact. The myelin loss-causing effect of KYNA occurred with no necrosis of oligodendrocytes, with locally severe astrogliosis and no cellular inflammatory response. Additionally, subdural KYNA infusion increased blood MOG concentration. Moreover, the rats infused with the highest doses of KYNA (1 and 10 nmol/min) demonstrated adverse neurological signs including weakness and quadriplegia.We suggest, that subdural infusion of high dose of KYNA can be used as an experimental tool for the study of mechanisms of myelin damage and regeneration. On the other hand, the administration of low, physiologically relevant doses of KYNA may help to discover the role of KYNA in control of physiological myelination process

The schematic presentation of intrathecal catheter placement.

<p>A–C7 –Th1 level of spinal cord, B–intrathecal catheter, C–a place of surgery.</p

Electron micrographs of severe demyelination in the area of the fasciculus gracilis of the dorsal column in the rat treated with the intrathecal infusion of 10 nmol/min of kynurenic acid (KYNA) per 5 days.

<p>A–in the area of severe demyelination, most of axons are naked, there are 3 astrocytes (As), one oligodendrocyte (OL) and a small blood vessel (bv). B–on higher magnification, the oligodendrocyte appears to have a compact cytoplasm devoid of processes; it is surrounded by many naked axons, some of the diameter greater than 2 μM (asterices) and a few myelinated axons. Size bars; A– 10 μM, B– 2 μM.</p

Electron micrographs of damaged myelin sheaths from the spinal cord of rats infused intrathecally with kynurenic acid (KYNA) for 7 days.

<p>A–an area from the dorsal column of a rat infused with 0.01 nmol/min of KYNA with an astrocyte (As) and an oligodendrocyte (OL) surrounded by damaged myelin sheaths. B–in this detail of A delineated by the white box, a segment of well compacted thick myelin sheath (Ms) passes into a segment were all lamellae are widely separated due to disintegration of compaction at the intraperiod line indicated by arrows. C–an example of a damaged myelin sheath from a single axon (Ax) from the lateral column of the rat infused with 10 nmol/min KYNA were a few well compacted lamellae (white double headed arrows) are widely separated by uncompacted lamellae (black arrows). D–in the lateral column of a rat infused with 1 nmol/min of KYNA, an axon (Ax) has a damaged myelin with segmental loss of compaction due to separation of lamellae at the intraperiod line (white arrow). There is a well-compacted thick myelin sheath (Ms) in the adjacent axon. E–lateral column from a rat infused with 0.0002 pmol/min of KYNA per 7 days, with multiple myelin sheaths showing the segmental loss of compaction and one oligodendrocyte (OL). The box indicates the area displayed in higher magnification in F–with two axons (Ax) surrounded by uncompacted myelin lamellae. Size bars; A, E– 5 μM, B, C, D– 100 nM, F– 1 μM.</p

Histomicrographs of the sections of the cervical spinal cord of rats infused intrathecally with kynurenic acid (KYNA).

<p>Toluidine blue (TB) stain. In the dorsal–A, and lateral–B, columns from a rat treated with 0.0002 pmol/min of KYNA per 7 days, there are sparse, individual axons with thickened myelin sheaths staining dark with TB. In the dorsal column–C of a rat infused with 0.01 nmol/min of KYNA per 7 days there are scattered myelin sheaths that are thickened, whereas in the lateral column–D, in the sub-pial areas, there are greater numbers of thickened myelin sheaths and there are also large axons that have dilated and attenuated myelin sheaths (arrows). The numbers of abnormal myelin sheaths appear to increase in the fasciculus gracilis (delineated by the arrow heads) and in the subpial areas of the dorsal and of the lateral and ventral columns in rats infused with; 0.1 nmol/min of KYNA per 7 days, E–dorsal column, F–lateral column; 1 nmol/min of KYNA per 7 days, G–dorsal column, H–ventral column; and 10 nmol/min of KYNA infused per 5 days, I–dorsal column, J–lateral column. Size bars– 50 μM.</p