Characterization of spinal subarachnoid bleeding associated to graded traumatic spinal cord injury in the rat.

STUDY DESIGN: Observational study in rats subjected to traumatic spinal cord injury (SCI). OBJECTIVES: To describe the features of spinal subarachnoid bleeding (SSB) occurring after graded SCI. SSB after SCI has been reported previously, but has not been studied systematically despite the fact that cerebral subarachnoid bleeding often produces severe neurological damage. SETTING: Mexico. METHODS: Anesthetized rats were subjected to mild or severe spinal cord contusion at T9. Occurrence, size, progression and location of SSB were characterized morphologically and scored from T7-T12 at 1 h and 1, 3 and 7 days post injury. Besides, contusions were videotaped to visualize bleeding at the moment of impact. RESULTS: SSB started immediately after contusion (severe or mild) and decreased gradually over time. For all vertebral segments, at all time points examined by histology, 48% of areas scored after severe contusion showed bleeding: 25% minor, 17% moderate and 6% major. After mild contusion, only 15% showed bleeding: 13 minor and 2% moderate. Maximum bleeding occurred early after injury in dorsal area of the epicenter in 100% of severe contusions (6% minor, 38 moderate and 56% major), and in 69% of mild contusions (63 minor and 6% moderate). CONCLUSION: Here, we detail SSB patterns occurring after graded SCI. Further studies are warranted to elucidate the possible role extramedullary events, such as SSB, in the pathophysiology of SCI that might encourage the development of new strategies for its management.

Lack of neuroprotection with pharmacological pretreatment in a paradigm for anticipated spinal cord lesions.

BACKGROUND: In humans elective spine surgery can cause iatrogenic spinal cord injury (SCI). Efforts for neuroprotection have been directed to avoid mechanical injury by using intraoperative monitoring and improving surgical techniques. There is, however, uncertainty regarding the efficacy of neuroprotective drugs. STUDY DESIGN: Experimental study on the effectiveness of pharmacological neuroprotection in an animal model of spine surgery simulating anticipated mechanically induced neurological damage. OBJECTIVE: To compare the efficacy of four drugs to protect against the neurological effects of iatrogenic SCI. SETTING: Research Unit for Neurological Diseases, IMSS-Proyecto Camina, Mexico City, Mexico. METHODS: Erythropoietin, melatonin, cyclosporine-A and methylprednisolone were administered to rats before, during and after controlled spinal cord contusion of mild intensity. Dosage was in accordance with their pharmacokinetic properties and experience gained with experimental SCI. Drug efficacy was assessed by motor function recovery over a period of 6 weeks and by spinal cord morphometry. RESULTS: Compared with animals treated with saline, the drug-treated groups showed no differences in their locomotor performance, nor in the amount of spared cord tissue. Notably, spontaneous activity was significantly reduced in rats treated with cyclosporine-A. CONCLUSION: The neuroprotectant drugs used here perioperatively did not reduce the extent of neurological damage in a model simulating iatrogenic SCI. Therefore, for now, the only protection in elective spine surgery is avoidance of primary injury altogether.

Acute spinal cord injury changes the disposition of some, but not all drugs given intravenously.

STUDY DESIGN: Experimental laboratory investigations in paraplegic rats. OBJECTIVE: In order to understand why acute spinal cord injury (SCI) changes the disposition of some, but not all drugs given intravenously (i.v.), pharmacokinetic parameters of drugs with different pharmacological properties were evaluated to determine the influence of SCI on physiological processes such as distribution, metabolism and excretion. SETTING: Mexico City, Mexico. METHODS: Rats were subjected to severe SCI (contusion) at T-9 level; pharmacokinetic studies of phenacetin, naproxen or gentamicin were performed 24 h after. These drugs were not chosen as markers because of their therapeutic properties, but because of their pharmacokinetic characteristics. Additional studies including plasma proteins, liver and renal function tests, and micro-vascular hepatic blood flow, were also performed at the same time after injury. RESULTS: Acute SCI significantly reduced distribution of drugs with intermediate and low binding to plasma proteins (phenacetin 30% and gentamicin 10%, respectively), but distribution did not change when naproxen - a drug highly bound to plasma proteins (99%) - was used, in absence of changes in plasma proteins. Metabolism was significantly altered only for a drug with liver blood flow - limited clearance (phenacetin) and not for a drug with liver capacity-limited clearance (naproxen). The liver function test did not change, whereas the hepatic micro-vascular blood flow significantly decreased after SCI. Renal excretion, evaluated by gentamicin clearance, was significantly reduced as a consequence of SCI, without significant changes in serum creatinine. CONCLUSIONS: Changes in drug disposition associated to acute SCI are complex and generalization is not possible. They are highly dependent on each drug properties as well as on the altered physiological processes. Results motivate the quest for strategies to improve disposition of selective i.v. drugs during spinal shock, in an effort to avoid therapeutic failure.

Molecular determinants of Akt-induced keratinocyte transformation.

The PI3K/PTEN/Akt signaling pathway has emerged in recent years as a main player in human cancers, increasing proliferation and decreasing apoptosis of transformed cells, and thus becoming a potential target for therapeutic intervention. Our previous data have demonstrated that Akt-mediated signaling is of a key relevance in the mouse skin carcinogenesis system, one of the best-known models of experimental carcinogenesis. Here, we investigated the involvement of several pathways as mediators of Akt-induced increased proliferation and tumorigenesis in keratinocytes. Tumors produced by subcutaneous injection of Akt-transformed keratinocytes showed increased Foxo3a phosphorylation, but no major alterations in p21(Cip1/WAF1), p27(Kip1) or mdm2 expression and/or localization. In contrast, we found increased expression and nuclear localization of DeltaNp63, beta-catenin and Lef1. Concomitantly, we also found increased expression of c-myc and CycD1, targets of the beta-catenin/Tcf pathway. Such increase is associated with increased phosphorylation and stabilization of c-myc protein as well as increased translation of c-myc and CycD1 due to mTOR activation. Using immunohistochemistry approaches in samples of oral dysplasias and human head and neck squamous cell carcinomas, we confirmed that increased Akt activation significantly correlates with increased DeltaNp63 and CycD expression, c-myc phosphorylation and nuclear accumulation of beta-catenin. Collectively, these results demonstrate that Akt is able to transform keratinocytes by specific mechanisms involving transcriptional and post-transcriptional processes.

Ajoene is an inhibitor and subversive substrate of human glutathione reductase and Trypanosoma cruzi trypanothione reductase: crystallographic, kinetic, and spectroscopic studies.

Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.