Efficacy and safety of lithium carbonate treatment of chronic spinal cord injuries: a double-blind, randomized, placebo-controlled clinical trial.

STUDY DESIGN: Lithium has attracted much attention as a neuroregenerative agent for spinal cord injury in animal models. We hypothesized that the lithium can be beneficial to patients with spinal cord injury. The safety and pharmacokinetics of lithium has been studied in our earlier phase I clinical trial, indicating its safety. This is a phase II clinical trial to evaluate its efficacy on chronic spinal cord injury patients. OBJECTIVES: The aim of this study was to investigate the efficacy of lithium on chronic spinal cord injury patients. SETTING: A major spinal cord injury rehabilitation center in Beijing, China. METHODS: Randomized, double-blind, placebo-controlled 6-week parallel treatment arms with lithium carbonate and with placebo. A total of 40 chronic spinal cord injury subjects were recruited. Oral lithium carbonate was titrated or placebo was simulated to maintain the serum lithium level of 0.6-1.2 mmol l(-1) for 6 weeks, followed by a 6-month follow-up. The functional outcomes and the neurological classifications, as well as the safety parameters, adverse events and pharmacokinetic data were carefully collected and monitored. RESULTS: No significant changes in the functional outcomes and the neurological classifications were found. The only significant differences were in the pain assessments using visual analog scale comparing the lithium and the placebo group. No severe adverse event was documented in the study. CONCLUSION: The lithium treatment did not change the neurological outcomes of patients with chronic spinal cord injury. It is worth to investigate whether lithium is effective in the treatment of neuropathic pain in chronic spinal cord injury. SPONSORSHIP: China Spinal Cord Injury Network Company Limited.

Neuroprotective effects of minocycline on double-stranded RNA-induced neurotoxicity in cultured cortical neurons.

1. Minocycline, memantine,and glycoconjugate were assessed for their ability to protect cultured primary cortical neurons against double-stranded RNA-induced neurotoxicity. 2. Minocycline but not memantine or glycoconjugate protected cultured cells and warrants further investigation.

A three-month, open-label, single-arm trial evaluating the safety and pharmacokinetics of oral lithium in patients with chronic spinal cord injury.

OBJECTIVES: Lithium has recently been found to enhance neuronal regeneration and differentiation. This arouses its potential use to treat spinal cord injury patients. The safety and pharmacokinetics of lithium are not verified for this group of patients as their internal organ functions may change. This is a phase 1 clinical trial to evaluate the safety and pharmacokinetics of lithium in spinal cord injury patients. METHODS: A total of 20 chronic spinal cord injury subjects were recruited. Oral lithium carbonate was given in divided dose to maintain the serum lithium level 0.6-1.2 mmol l(-1) for 6 weeks. Safety parameters, adverse events and pharmacokinetic data were carefully collected and monitored. RESULTS: No severe adverse event was documented. All blood parameters remained stable. Nausea and vomiting were the most common complaints but tolerance was improved in 2 weeks for most subjects. A wide range of oral doses was required to maintain serum lithium level at the targeted range. However, the dose for individual subject was relatively constant. CONCLUSION: This phase 1 clinical trial is the first report indicating the safety of lithium in chronic spinal cord injury patients. It is well tolerated after the first 2 weeks. Individual titration of lithium is essential to maintain an optimal serum lithium level but once the desirable level is achieved, the oral dose remains relatively unchanged for maintenance.

Oxygen-derived free radicals mediate endothelium-dependent contractions in femoral arteries of rats with streptozotocin-induced diabetes.

BACKGROUND AND PURPOSE: The present experiments were designed to study the contribution of oxygen-derived free radicals to endothelium-dependent contractions in femoral arteries of rats with streptozotocin-induced diabetes. EXPERIMENTAL APPROACH: Rings with and without endothelium were suspended in organ chambers for isometric tension recording. The production of oxygen-derived free radicals in the endothelium was measured with 2',7'-dichlorodihydrofluorescein diacetate using confocal microscopy. The presence of protein was measured by western blotting. KEY RESULTS: In the presence of L-NAME, the calcium ionophore A23187 induced larger endothelium-dependent contractions in femoral arteries from diabetic rats. Tiron, catalase, deferoxamine and MnTMPyP, but not superoxide dismutase reduced the response, suggesting that oxygen-derived free radicals are involved in the endothelium-dependent contraction. In the presence of L-NAME, A23187 increased the fluorescence signal in femoral arteries from streptozotocin-treated, but not in those from control rats, confirming that the production of oxygen-derived free radicals contributes to the enhanced endothelium-dependent contractions in diabetes. Exogenous H2O2 caused contractions in femoral arterial rings without endothelium which were reduced by deferoxamine, indicating that hydroxyl radicals contract vascular smooth muscle and thus could be an endothelium-derived contracting factor in diabetes. The reduced presence of Mn-SOD and the decreased activity of catalase in femoral arteries from streptozotocin-treated rats demonstrated the presence of a redox abnormality in arteries from rats with diabetes. CONCLUSIONS AND IMPLICATIONS: These findings suggest that the redox abnormality resulting from diabetes increases oxidative stress which facilitates and/or causes endothelium-dependent contractions.

Calcium and reactive oxygen species increase in endothelial cells in response to releasers of endothelium-derived contracting factor.

BACKGROUND AND PURPOSE: Experiments were designed to assess whether or not the intracellular concentration of calcium and reactive oxygen species (ROS) increase in endothelial cells of the rat thoracic aorta in response to releasers of endothelium-derived contracting factor (EDCF) and if so, whether or not a difference exists between spontaneously hypertensive (SHR) and normotensive (WKY) rats. EXPERIMENTAL APPROACH: Calcium and ROS were measured by confocal microscopy, using Fura-red in combination with Fluo-4 and dichlorodihydrofluorescein diacetate, respectively. KEY RESULTS: Acetylcholine caused a rapid increase in cytosolic calcium concentration in endothelial cells of both SHR and WKY, which was significantly more pronounced in aortae of the former strain. This rise of calcium was not affected by indomethacin (an inhibitor of cyclooxygenase) or Tiron plus diethyldithiocarbamate acid (DETCA) (membrane permeable antioxidants). In the presence of a nitric oxide synthase blocker, acetylcholine also caused a rapid increase in ROS in endothelial cells of SHR but not in those of WKY. The burst of ROS was prevented by indomethacin or Tiron plus DETCA. CONCLUSIONS AND IMPLICATIONS: These experiments show that endothelial cells of SHR are more prone to calcium and ROS overload upon stimulation with acetylcholine. The abnormal accumulation of calcium is a prerequisite to initiate the release of EDCF and can be mimicked using the calcium ionophore A23187. The sequence of events occurring during endothelium-dependent contractions firstly requires the accumulation of calcium, which then activates cyclooxygenase and produces ROS along with EDCF that in turn stimulates TP-receptors, resulting in EDCF-mediated contractions.

Expression of telomerase reverse transcriptase in adult goldfish retina.

Telomerase, a specialized reverse transcriptase that maintains telomere during cell division, is commonly associated with cell proliferation. Increasing evidence suggests that telomerase may bear functions other than telomere elongation. We investigated whether telomerase is expressed in the continuously growing goldfish retina. Telomeric repeat amplification protocol (TRAP) assay reveals telomerase activity in goldfish retina. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot show that telomerase catalytic subunit (TERT) is expressed at both mRNA and protein levels. Localization of TERT by immunohistochemistry indicates prominent expression of TERT in the outer nuclear layer, the inner nuclear layer, and, in a small population of cells, in the ganglion cell layer. Coexpression of TERT with proliferative cell nuclear antigen (PCNA) immunoreactivity is found in rod progenitor cells. These results suggest the role of telomerase in vertebrate central nervous system (CNS) other than telomere maintenance, such as regulation of cell cycle progression and maintenance of retinal cell phenotypes.

Photochemical cross-linking for collagen-based scaffolds: a study on optical properties, mechanical properties, stability, and hematocompatibility.

Collagen presents an attractive biomaterial for tissue engineering because of its excellent biocompatibility and negligible immunogenicity. However, some intrinsic features related to the mechanical stability and thrombogenicity limit its applications in orthopedic and vascular tissue engineering. Photochemical cross-linking is an emerging technique able to stabilize tissue grafts and improve the physicochemical properties of collagen-based structures. However, other important properties of collagen-based structures and the effect of processing parameters on these properties have not been explored. In this study, we aim to investigate the dose dependence of tensile and swelling properties on two parameters, namely, laser energy fluence and rose Bengal photosensitizer concentration. We also study the compression properties using cyclic compression test, long-term stability using subcutaneous implantation, and hematocompatibility using platelets adhesion test, of cross-linked collagen structures. Moreover, because limited optical penetration in turbid media is the major obstacle for light-based techniques, we also characterize the optical properties, which partially determine the effective optical penetration depth in collagen gel samples, during photochemical cross-linking. Laser energy fluence and rose Bengal concentration are important parameters affecting the cross-linking efficiency, which was characterized as the mechanical and the swelling properties, in a dose-dependent manner. Under the experimental conditions in this study, the peak fluence was 12.5 J/cm2 and the minimal rose Bengal concentration for effective cross-linking was >0.00008% (0.786 micromol). Photochemical cross-linking also enhanced the compression strength and long-term stability of collagen structures without compromising the tissue compatibility. Furthermore, photochemical cross-linking reduced platelet adhesion and abolished fibrin mesh formation, thereby improving the hematocompatibility of collagen structures. These results suggest the feasibility of using the photochemically cross-linked collagen structures for orthopedic and vascular tissue engineering. Finally, the effective optical penetration depth in collagen gel samples is wavelength and rose Bengal concentration dependent, and was approximately 12 mm at 514 nm at 0.001% (9.825 micromol), the rose Bengal concentration mostly used in this study.

Treadmill exercise suppressed stress-induced dendritic spine elimination in mouse barrel cortex and improved working memory via BDNF/TrkB pathway.

Stress-related memory deficit is correlated with dendritic spine loss. Physical exercise improves memory function and promotes spinogenesis. However, no studies have been performed to directly observe exercise-related effects on spine dynamics, in association with memory function. This study utilized transcranial two-photon in vivo microscopy to investigate dendritic spine formation and elimination in barrel cortex of mice under physical constrain or naive conditions, followed by memory performance in a whisker-dependent novel texture discrimination task. We found that stressed mice had elevated spine elimination rate in mouse barrel cortex plus deficits in memory retrieval, both of which can be rescued by chronic exercise on treadmill. Exercise also elevated brain-derived neurotrophic factor (BDNF) expression in barrel cortex. The above-mentioned rescuing effects for both spinognesis and memory function were abolished after inhibiting BDNF/tyrosine kinase B (TrkB) pathway. In summary, this study demonstrated the improvement of stress-associated memory function by exercise via facilitating spine retention in a BDNF/TrkB-dependent manner.

Retinal ganglion cells toxicity caused by photosensitising effects of intravitreal indocyanine green with illumination in rat eyes.

AIM: To investigate the effects of indocyanine green (ICG) with or without illumination on rat retinal ganglion cells (RGC) and retinal morphology. METHODS: Intravitreal injections of 1.0 mg/ml ICG solution were performed in rat eyes with or without subsequent illumination for 5 minutes. Eyes in the control group had intravitreal injections of balanced salt solution with illumination. Retrograde labelling of RGC with 6% Fluoro-Gold was performed 1 month later and RGC densities were compared between the three groups. Light microscopy with measurements of outer nuclear layer (ONL) and inner nuclear layer (INL) thicknesses were also performed and compared. RESULTS: Eyes with ICG without illumination showed insignificant reduction in RGC density compared with the control group (p = 0.28), whereas a significant decrease in RGC density was found in eyes that had ICG injection with illumination (p = 0.036). A significant increase in ONL thickness was also observed in the ICG with illumination treated eyes compared with the ICG without illumination and the control groups (p<0.001). No significant difference in INL thickness was observed between the three groups. CONCLUSIONS: Intravitreal injection of 0.1 mg/ml ICG in rat eyes followed by illumination resulted in photosensitising toxicity to RGC. Lower ICG concentration or illumination level should be considered when performing ICG assisted macular surgery.

Anxiety and depression with neurogenesis defects in exchange protein directly activated by cAMP 2-deficient mice are ameliorated by a selective serotonin reuptake inhibitor, Prozac.

Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1(-/-)) or Epac2 (Epac2(-/-)) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2(-/-) mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2(-/-) mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2(-/-) mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis.

Axonal regeneration of retinal ganglion cells: effect of trophic factors.

A variety of neurotrophic factors can influence the cell functions of the developing, mature and injured retinal ganglion cells. The discovery that retinal ganglion cell loss can be alleviated by neurotrophic factors has generated a great deal of interest in the therapeutic potential of these molecules. Recently, evidence has provided valuable information on the receptors that mediate these events and the intracellular signaling cascades after the binding of these ligands. Signaling by neurotrophic factors does not seem to restrict to retrograde messenger from the target but also includes local interactions with neighbouring cells along the axonal pathways, anterograde signaling from the afferents and autocrine signaling. More insight into the mechanisms of action of neurotrophic factors and the signal transduction pathway leading to the protection and regeneration of retinal ganglion cells may allow the design of new therapeutic strategies.

Photochemical crosslinking improves the physicochemical properties of collagen scaffolds.

Collagen is a natural biomaterial with excellent biocompatibility. However, unprocessed collagen has low stability and weak mechanical strength, which limits its application in tissue engineering. The current study aimed to improve the physicochemical properties of collagen scaffolds by using photochemical crosslinking. Collagen gel was reconstituted and photochemically crosslinked by using laser irradiation in the presence of a photosensitizer. Scanning electron microscope was used to characterize the surface and cross-sectional morphology. Stress-strain relationship and other mechanical properties were determined by uniaxial tensile tests. Thermostability and water-binding capacities also were analyzed by using differential scanning calorimetry and swelling ratio measurements, respectively. Photochemically crosslinked porous structures showed fine microstructure with interconnected micron-sized pores, whereas uncrosslinked controls only showed macrosheet-like structures. The stabilizing effect of photochemical crosslinking also was revealed by retaining the three-dimensional lamellae-like structures after thermal analysis in crosslinked membranes but not in the controls. Photochemical crosslinking also significantly reduced the swelling ratio, improved the stress-strain relationship, peak load, ultimate stress, rupture strain, and tangent modulus of collagen membranes. The current study showed that an innovative photochemical crosslinking process was able to produce collagen scaffolds with fine microstructures; to strengthen, stiffen, and stabilize collagen membranes; and to modify their swelling ratio. This may broaden the use of collagen-based scaffolds in tissue engineering, particularly for weight-bearing tissues.

The injury resistant ability of melanopsin-expressing intrinsically photosensitive retinal ganglion cells.

Neurons in the mammalian retina expressing the photopigment melanopsin have been identified as a class of intrinsically photosensitive retinal ganglion cells (ipRGCs). This discovery more than a decade ago has opened up an exciting new field of retinal research, and following the initial identification of photosensitive ganglion cells, several subtypes have been described. A number of studies have shown that ipRGCs subserve photoentrainment of circadian rhythms. They also influence other non-image forming functions of the visual system, such as the pupillary light reflex, sleep, cognition, mood, light aversion and development of the retina. These novel photosensitive neurons also influence form vision by contributing to contrast detection. Furthermore, studies have shown that ipRGCs are more injury-resistant following optic nerve injury, in animal models of glaucoma, and in patients with mitochondrial optic neuropathies, i.e., Leber's hereditary optic neuropathy and dominant optic atrophy. There is also an indication that these cells may be resistant to glutamate-induced excitotoxicity. Herein we provide an overview of ipRGCs and discuss the injury-resistant character of these neurons under certain pathological and experimental conditions.

Diffusivity of the uncinate fasciculus in heroin users relates to their levels of anxiety.

Heroin use is closely associated with emotional dysregulation, which may explain its high comorbidity with disorders such as anxiety and depression. However, the understanding of the neurobiological etiology of the association between heroin use and emotional dysregulation is limited. Previous studies have suggested an impact of heroin on diffusivity in white matter involving the emotional regulatory system, but the specificity of this finding remains to be determined. Therefore, this study investigated the association between heroin use and diffusivity of white matter tracts in heroin users and examined whether the tracts were associated with their elevated anxiety and depression levels. A sample of 26 right-handed male abstinent heroin users (25 to 42 years of age) and 32 matched healthy controls (19 to 55 years of age) was recruited for this study. Diffusion tensor imaging data were collected, and their levels of anxiety and depression were assessed using the Hospital Anxiety and Depression Scale. Our findings indicated that heroin users exhibited higher levels of anxiety and depression, but the heroin use-associated left uncinate fasciculus was only related to their anxiety level, suggesting that association between heroin and anxiety has an incremental organic basis but that for depression could be a threshold issue. This finding improves our understanding of heroin addiction and its comorbid affective disorder and facilitates future therapeutic development.

Development of abnormal recrossing retinotectal projections after superior colliculus lesions in newborn Syrian hamsters.

The development of the retinal projections to the roof of the midbrain was studied in Syrian hamsters after right superior colliculus (SC) lesions on the day of birth, using both autoradiographic and degeneration techniques. The dead tissue resulting from the heat lesion is not completely removed until the eighth day after birth. Normally the midline of the SC is defined by a pia-lined fissure separating the left and right colliculi, but in the animals with early unilateral lesions, the pia at the midline is damaged. When it regrows, together with vascular and other meningeal tissues, it forms a flat tissue bridge across the midline as early as two days after the lesion. When the axons from the left eye reach the right SC, they encounter the dead tissue and separate into two bundles. One bundle courses over the surface of the dead tissue and one grows underneath it. It is not until the third to fourth day that axons in the dorsal bundle cross the midline, via the tissue bridge, to terminate anomalously in the medial wall of the left SC. When the quantity of such recrossing axons is small, they overlap extensively with the optic tract fibers from the other (right) eye which normally have innervated the entire SC by day 3. However, it appears that as the density of the recrossing axons increases they displace the axons originating in the other eye from the medial wall of the left SC. Thus, eventually fibers from both eyes terminate in the left SC, occupying separate territories with little, if any, overlap. Axons in the ventral bundle begin to innervate the deep layers of the right SC on day 2. These axons were never observed to recross the midline. These results indicate that mechanical guidance and axonal segregation dependent on relative densities are two processes that govern the development of retinotectal projections after early SC lesions in hamsters.

Effects of visual or light deprivation on the morphology, and the elimination of the transient features during development, of type I retinal ganglion cells in hamsters.

Intracellular injection of Lucifer Yellow (LY) was used to study the detailed morphology of the normal visually deprived, and light-deprived superior colliculus projecting Type I retinal ganglion cells (RGCs) in hamsters. The soma size of the normal Type I cells ranged from 337 to 583 microns 2 with a mean of 436 microns 2. Two to six primary dendrites were observed in these cells. The mean dendritic field diameter was 495 microns and ranged from 309 to 702 microns. The dendritic field diameter of this population of cells exhibited an eccentricity dependence. Quantitative comparisons between the normal and visually deprived or light-deprived Type I RGCs indicated that the morphology of these three groups of cells were similar to each other in terms of the soma size, dendritic field diameter, branching pattern, and total length of the dendrites. During the normal development of cats and hamsters, several transient features, such as exuberant dendritic spines and intraretinal axonal branches, have been observed in the developing RGCs. The complete elimination of these transient features occurs at about 3 and 2 weeks after the opening of the eyes in cats and hamsters, respectively. In the present study, the hypothesis whether visual experience or light stimulation is required for the elimination of these transient features during development was examined. After studying a total of 115 mature Type I RGCs, which included cells from the normal, visually deprived and light deprived animals, no transient feature was observed. We conclude that visual or light deprivation has no effect on the morphological development of superior colliculus projecting Type I RGCs in hamsters, and the elimination of the transient features on the Type I RGCs during development does not depend on visual experience or light stimulation.

Postnatal development of type I retinal ganglion cells in hamsters: a lucifer yellow study.

The postnatal development of a population of superior colliculus projecting retinal ganglion cells with large somata in hamsters aged from postnatal day (P) 4 to adult was studied by the intracellular injection of Lucifer Yellow. This population of cells was interpreted as Type I cells based on their large soma sizes and dendritic morphology resembling that of mature Type I cells. In addition to the growth of the soma and the dendritic field, transient morphological features such as intraretinal axon collaterals and exuberant dendritic spines, but not somatic spines, were frequently observed on this population of cells in hamsters during development. None of them exhibited any intraretinal axon collaterals after P7. The number of transient spine-like processes on dendrites increased from P4 onwards to reach a peak at P16, decreased abruptly within a few days after the peak, and stabilised to reach the adult level by P30. These developing cells attained the maximum number of dendritic branches by P16 and there seems to be little, if any, reduction in the number of branch points after this time point. In addition, the length of individual branches of dendrites was not increased excessively during development and then shortened during maturation. Thus, the dendritic remodeling of these cells after P16 seems to be mainly the increase of the length of dendrites and the removal of exuberant dendritic spines.

NADPH-diaphorase neurons in the retina of the hamster.

NADPH-diaphorase-positive neurons have been demonstrated in the inner nuclear layer and ganglion cell layer of the retina of different mammalian species, but so far no experiments have been conducted to identify whether these cells are amacrine cells and/or retinal ganglion cells. We attempted to solve this problem by studying the NADPH-diaphorase-positive neurons in the hamster retina. From the NADPH-diaphorase histochemical reaction, two distinct types of neurons in the hamster retina were identified. They were named ND(g) and ND(i) cells. The ND(g) cells were cells with larger somata, ranging from 10 to 21 microns in diameter with a mean of 15.58 microns (S.D. = 2.59). They were found in the ganglion cell layer only. The ND(i) cells were smaller, with the somata ranging from 7 to 11 microns and having the mean diameter of 8.77 microns (S.D. = 1.24). Most of the ND(i) cells were found in the inner nuclear layer, and only very few could be observed in the inner plexiform layer. On average, there were 8,033 ND(g) and 5,051 ND(i) cells in the ganglion cell layer and inner nuclear layer, respectively. Two experiments were performed to clarify whether any of the NADPH-diaphorase neurons were retinal ganglion cells. Following unilateral optic nerve section, which leads to the retrograde degeneration of retinal ganglion cells, the numbers of both ND(g) and ND(i) cells did not change significantly for up to 4 months. In addition, when retinal ganglion cells were prelabeled retrogradely (horseradish peroxidase or fluorescent microspheres) and retinas were then stained for NADPH diaphorase, no double-labeled neurons were detected. These results indicated that the NADPH-diaphorase neurons in the hamster retina were the amacrine cells in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. Dendrites of the ND(g) and ND(i) cells were found to stratify in sublaminae 1, 3, and 5 of the inner plexiform layer, with a prominent staining in the sublamina 5. The possible importance of this arrangement in the rod pathway is also discussed.