Tamoxifen promotes differentiation of oligodendrocyte progenitors in vitro.

The most promising therapeutic approach to finding the cure for devastating demyelinating conditions is the identification of clinically safe pharmacological agents that can promote differentiation of endogenous oligodendrocyte precursor cells (OPCs). Here we show that the breast cancer medication tamoxifen (TMX), with well-documented clinical safety and confirmed beneficial effects in various models of demyelinating conditions, stimulates differentiation of rat glial progenitors to mature oligodendrocytes in vitro. Clinically applicable doses of TMX significantly increased both the number of CNPase-positive oligodendrocytes and protein levels of myelin basic protein, measured with Western blots. Furthermore, we also found that OPC differentiation was stimulated, not only by the pro-drug TMX-citrate (TMXC), but also by two main TMX metabolites, 4-hydroxy-TMX and endoxifen. Differentiating effects of TMXC and its metabolites were completely abolished in the presence of estrogen receptor (ER) antagonist, ICI182780. In contrast to TMXC and 4-hydroxy-TMX, endoxifen also induced astrogliogenesis, but independent of the ER activation. In sum, we showed that the TMX prodrug and its two main metabolites (4-hydroxy-TMX and endoxifen) promote ER-dependent oligodendrogenesis in vitro, not reported before. Given that differentiating effects of TMX were achieved with clinically safe doses, TMX is likely one of the most promising FDA-approved drugs for the possible treatment of demyelinating diseases.

Multiple sclerosis and breast cancer.

Multiple sclerosis (MS) and breast cancer (BC) share common features; most notably, both are more frequent in women than in men. In addition to the involvement of sex hormones, a number of genetic and pharmacological studies support a possible relationship between these two diseases. However, there are no conclusive epidemiological findings related to MS and BC worldwide, and there are no recent data for the US population. We conducted a case-control study using a hospital inpatient discharge dataset (21,536 cases and two control series totaling 59,581 controls) from the Texas Health Care Information Collection. We assessed occurrence of MS in BC cases and in two control series: diabetes mellitus type II, and open wounds. After controlling for age, race-ethnicity, and health insurance status, a statistically-significant protective association was detected: BC cases were 45% less likely than diabetic controls to have MS (OR=0.55, 95% CI=0.37-0.81), and 63% less likely than open wound controls to have MS (OR=0.37, 95% CI=0.21-0.66). Our study presented here is the only current assessment of the association between MS and BC in the USA and suggests a protective effect of MS on BC in the hospitalized population.

Induction of angiopoietin-2 after spinal cord injury.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have opposing effects on blood vessels, with Ang-2 being mainly induced during the endothelial barrier breakdown. It is known that spinal cord injury (SCI) induces lasting decreases in Ang-1 levels, underlying endothelial barrier disruption, but the expression of Ang-2 in spinal cord injury has not been studied. We characterized Ang-2 after SCI using a clinically relevant rat model of contusion SCI. We found that SCI induces marked and persistent upregulation of Ang-2 (up to 10 weeks after SCI), which does not reflect well-characterized temporal profile of the blood-spinal cord barrier (BSCB) breakdown after SCI, and thus suggests other role(s) for Ang-2 in injured spinal cords. Furthermore, we also found that higher Ang-2 levels were associated with more successful locomotor recovery after SCI, both in SCI rats with markedly better spontaneous motor recovery and in SCI rats receiving a neuroprotective pharmacological intervention (amiloride), suggesting a beneficial role for Ang-2 in injured spinal cords. Immunocytochemical analyses revealed that Ang-2 was not induced in endothelial cells, but in perivascular and non-vascular cells labeled with glial fibrillary acidic protein (GFAP) or with chondroitin sulfate proteoglycan (NG2). Therefore, it is unlikely that induction of Ang-2 contributes to vascular dysfunction underlying functional impairment after SCI, but rather that it contributes to the beneficial pro-angiogenic and/or gliogenic processes underlying recovery processes after SCI.

Aquaporins in spinal cord injury: the janus face of aquaporin 4.

Although malfunction of spinal cord water channels (aquaporins, AQP) likely contributes to severe disturbances in ion/water homeostasis after spinal cord injury (SCI), their roles are still poorly understood. Here we report and discuss the potential significance of changes in the AQP4 expression in human SCI that generates glial fibrillary acidic protein (GFAP)-labeled astrocytes devoid of AQP4, and GFAP-labeled astroglia that overexpress AQP4. We used a rat model of contusion SCI to study observed changes in human SCI. AQP4-negative astrocytes are likely generated during the process of SCI-induced replacement of lost astrocytes, but their origin and role in SCI remains to be investigated. We found that AQP4-overexpression is likely triggered by hypoxia. Our transcriptional profiling of injured rat cords suggests that elevated AQP4-mediated water influx accompanies increased uptake of chloride and potassium ions which represents a protective astrocytic reaction to hypoxia. However, unbalanced water intake also results in astrocytic swelling that can contribute to motor impairment, but likely only in milder injuries. In severe rat SCI, a low abundance of AQP4-overexpressing astrocytes was found during the motor recovery phase. Our results suggest that severe rat contusion SCI is a better model to analyze AQP4 functions after SCI. We found that AQP4 increases in the chronic post-injury phase are associated with the development of pain-like behavior in SCI rats, while possible mechanisms underlying pain development may involve astrocytic swelling-induced glutamate release. In contrast, the formation and size of fluid-filled cavities occurring later after SCI does not appear to be affected by the extent of increased AQP4 levels. Therefore, the effect of therapeutic interventions targeting AQP4 will depend not only on the time interval after SCI or animal models, but also on the balance between protective role of increased AQP4 in hypoxia and deleterious effects of ongoing astrocytic swelling.

Aquaporin 1 - a novel player in spinal cord injury.

The role of water channel aquaporin 1 (AQP-1) in uninjured or injured spinal cords is unknown. AQP-1 is weakly expressed in neurons and gray matter astrocytes, and more so in white matter astrocytes in uninjured spinal cords, a novel finding. As reported before, AQP-1 is also present in ependymal cells, but most abundantly in small diameter sensory fibers of the dorsal horn. Rat contusion spinal cord injury (SCI) induced persistent and significant four- to eightfold increases in AQP-1 levels at the site of injury (T10) persisting up to 11 months post-contusion, a novel finding. Delayed AQP-1 increases were also found in cervical and lumbar segments, suggesting the spreading of AQP-1 changes over time after SCI. Given that the antioxidant melatonin significantly decreased SCI-induced AQP-1 increases and that hypoxia inducible factor-1alpha was increased in acutely and chronically injured spinal cords, we propose that chronic hypoxia contributes to persistent AQP-1 increases after SCI. Interestingly; AQP-1 levels were not affected by long-lasting hypertonicity that significantly increased astrocytic AQP-4, suggesting that the primary role of AQP-1 is not regulating isotonicity in spinal cords. Based on our results we propose possible novel roles for AQP-1 in the injured spinal cords: (i) in neuronal and astrocytic swelling, as AQP-1 was increased in all surviving neurons and reactive astrocytes after SCI and (ii) in the development of the neuropathic pain after SCI. We have shown that decreased AQP-1 in melatonin-treated SCI rats correlated with decreased AQP-1 immunolabeling in the dorsal horns sensory afferents, and with significantly decreased mechanical allodynia, suggesting a possible link between AQP-1 and chronic neuropathic pain after SCI.

Acute and chronic changes in aquaporin 4 expression after spinal cord injury.

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.

Statistical approach to DNA chip analysis.

Statistical methods for analyzing data from DNA microarray experiments are reviewed. Specifically, we discuss common experimental setups, methods for data reduction and clustering, and analysis of time-course experiments. While early microarray studies focused mainly on the basic methodological and technical aspects of DNA arrays, emphasis has shifted to biological, medical, and clinical applications. We mention several of these and present results from our recent research as illustrative examples. New developments in this ever-growing field are outlined.

DNA microarray analysis of the contused spinal cord: effect of NMDA receptor inhibition.

Spinal cord injury (SCI)-induced neurodegeneration leads to irreversible and devastating motor and sensory dysfunction. Post-traumatic outcomes are determined by events occurring during the first 24 hours after SCI. An increase in extracellular glutamate concentration to neurotoxic levels is one of the earliest events after SCI. We used Affymetrix DNA oligonucleotide microarrays (with 1,322 DNA probes) analysis to measure gene expression in order to test the hypothesis that SCI-induced N-methyl-D-aspartate (NMDA) receptor activation triggers significant postinjury transcriptional changes. Here we report that SCI, 1 hour after trauma, induced change in mRNA levels of 165 genes and expression sequence tags (ESTs). SCI affected mRNA levels of those genes that regulate predominantly transcription factors, inflammation, cell survival, and membrane excitability. We also report that NMDA receptor inhibition (with -(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate [MK-801]) reversed the effect of SCI on about 50% of the SCI-affected mRNAs. Especially interesting is the finding that NMDA receptor activation participates in the up-regulation of inflammatory factors. Therefore, SCI-induced NMDA receptor activation is one of the dominant, early signals after trauma that leads to changes in mRNA levels of a number of genes relevant to recovery processes. The majority of MK-801 effects on the SCI-induced mRNA changes reported here are novel. Additionally, we found that the MK-801 treatment also changed the mRNA levels of 168 genes and ESTs that had not been affected by SCI alone, and that some of their gene products could have harmful effects on SCI outcome.

Bcl-xL expression after contusion to the rat spinal cord.

After contusion-derived spinal cord injury, (SCI) there is localized tissue disruption and energy failure that results in early necrosis and delayed apoptosis, events that contribute to chronic central pain in a majority of patients. We assessed the extent of contusion-induced apoptosis of neurons in a known central pain-signaling pathway, the spinothalamic tract (STT), which may be a contributor to SCI-induced pain. We observed the loss of STT cells and localized increase of DNA fragmentation and cytoplasmic histone-DNA complexes, which suggested potential apoptotic changes among STT neurons after SCI. We also showed SCI-associated changes in the expression of the antiapoptotic protein Bcl-xL, especially among STT cells, consistent with the hypothesis that Bcl-xL regulates the extent of apoptosis after SCI. Apoptosis in the injured spinal cord correlated well with prompt decreases in Bcl-xL protein levels and Bcl-xL/Bax protein ratios at the contusion site. We interpret these results as evidence that regulation of Bcl-xL may play a role in neural sparing after spinal injury and pain-signaling function.

Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound.

The use of systemic IGF-1 has been shown to attenuate the postburn hypermetabolic response and improve burn wound healing. Local IGF-1 gene therapy, however, promotes re-epithelialization in the burn wound without the side-effects associated with systemic delivery. We tested the hypothesis that these beneficial effects are due to changes in local cytokine production. Adult male Sprague-Dawley rats received a 40% total body surface area full-thickness scald burn and randomly received a subcutaneous injection at the burn wound margin of saline or cationic liposomes containing a IGF-1 cDNA construct. Animals were killed at 1, 4, 7 and 10 days after burn trauma. Skin biopsies at the wound border were harvested for total RNA extraction. Cytokine mRNA expression was determined using a multi-probe RNase protection assay. Data are presented as means +/- s.e.m. Statistical analysis used the unpaired t-test or Mann-Whitney test where appropriate. Significance was accepted at P < 0.05. Treatment of the burn wound with liposomal IGF-1-cDNA transfer decreased IL-1beta mRNA levels on day 10 after burn trauma from five-fold burn-induced increases compared with sham-treated rats, to near the control values present in unburned skin samples. Similarly, there was an eight-fold increase in TNF-alpha mRNA expression on postburn day 10 that was abrogated by IGF-1 gene therapy. Local IGF-1 gene transfer attenuates the mRNA expression of the inflammatory cytokines IL-1beta and TNF-alpha in the burn wound. This change may improve burn wound healing by decreasing prolonged local inflammation.

IL-1 receptor antagonist prevents apoptosis and caspase-3 activation after spinal cord injury.

One of the consequences of cytokine-orchestrated inflammation after CNS trauma is apoptosis. Our hypothesis is that cell death in the spinal cord after injury results in part from increased synthesis and release of IL-1beta. Using a ribonuclease protection assay, we demonstrated that there is increased transient expression of IL-1beta mRNA and, by using IL-1beta protein ELISA assay, that there are increased IL-1beta protein levels in the contused rat spinal cord, initially localized to the impact region of the spinal cord (segment T8). Using an ELISA cell death assay, we showed that there is apoptosis in the spinal cord 72 h after injury, a finding that was confirmed by measuring caspase-3 activity, which also significantly increased at the site of injury 72 h after trauma. Treatment of the contused spinal cord at the site of injury with the IL-1 receptor antagonist (rmIL-lra, 750 ng/mL) for 72 h using an osmotic minipump completely abolished the increases in contusion-induced apoptosis and caspase-3 activity.

Glutamate as a putative neurotransmitter in the mollusc, Lymnaea stagnalis.

Bath-applied glutamate (10-1000 microM) produced excitatory and inhibitory responses on numerous identified neurons of the mollusc Lymnaea stagnalis. Using both in situ and in vitro preparations, glutamate or glutamate agonists produced a depolarization in identified neurons right pedal dorsal 1 and right pedal dorsal 2 and 3. However, attempts to block glutamate-evoked responses with glutamate antagonists were unsuccessful. We examined a potential glutamatergic neuron, visceral dorsal 4. Exogenous application of the peptides (GDPFLRFamide and SDPFLRFamide) could mimic the inhibitory, but not the excitatory effects of visceral dorsal 4 on its postsynaptic cells, implying the presence of a second transmitter. We tested the possibility that glutamate is this second neurotransmitter by using excitatory synapses between visceral dorsal 4 and postsynaptic cells right pedal dorsal 2 and 3, right pedal dorsal 1, visceral F group and right parietal B group neurons. Of all the putative neurotransmitters tested, only glutamate had consistent excitatory effects on these postsynaptic cells. Also, the amplitude of the right pedal dorsal 2 and 3 excitatory postsynaptic potentials was reduced in the presence of N-methyl-D-aspartate and other glutamate agonists, suggesting desensitization of the endogenous transmitter receptor. In conclusion, some identified Lymnaea neurons respond to glutamate via a receptor with novel pharmacological properties. Furthermore, a Lymnaea interneuron may employ glutamate as a transmitter at excitatory synapses.

Characteristics of outward current induced by application of dopamine on a snail neuron.

1. A closer characterization of the potassium channel opened by the application of dopamine (DA) on an identified Helix pomatia neuron was attempted. The effect of K+ channel blockers (TEA and 4-AP) on the DA-induced current was examined. The results indicate that the channel opened by DA does not share the pharmacological properties of other snail neuron K-channels. 2. The I-V relation for IDA was successfully fitted by the Constant Field equation except below the reversal potential where the current was smaller than expected. The assumption that DA binding is voltage-sensitive is supported by the increment of the Hill coefficient with hyperpolarization (from nH approximately equal to 1 to nH approximately equal to 2). 3. The presence of the phosphodiesterase inhibitor IBMX does not affect the DA induced outward current. However, the assumption that the snail neurons' DA receptor belongs to the D2 class is in contrast to the antagonistic effects of ergot alkaloids which, in mammalian neurons, are competitive antagonists of D1 receptors. 4. The examination of the voltage-sensitivity of the blocking action of the ergot alkaloid (Bromoergocryptinine) revealed that it does not compete with DA for the same binding site as in mammalian D1 receptors.