RESUMEN
A cationic lipid-based gene delivery system composed of N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 microg of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.
Asunto(s)
Carcinoma de Células Escamosas/terapia , Terapia Genética , Lípidos/administración & dosificación , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/patología , Cationes , División Celular , Cartilla de ADN , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C3H , Plásmidos , TransfecciónRESUMEN
BACKGROUND AND PURPOSE: This study explores the temporal relation of the severity of ischemia and calcium-calmodulin binding in vulnerable and resistant brain regions in a commonly used model of global ischemia. METHODS: Immunohistochemical assay of free calmodulin unbound to calcium and light microscopic histological damage were measured in rats after 5, 10, or 20 minutes of global ischemia. RESULTS: After 24 hours of reperfusion, decreased calmodulin staining, representing increased calcium influx and calcium-calmodulin binding, correlated with increasing durations of ischemia across all brain regions. Based on a 4-point scale (4, extensive stain; 0, no staining), calmodulin staining after 5 minutes versus 10 minutes of ischemia was 3.2 versus 1.9, respectively (p less than 0.05) and after 10 minutes versus 20 minutes of ischemia was 1.9 versus 1.0, respectively (p less than 0.01). The CA1 region displayed the greatest sensitivity to ischemia. Similar but less dramatic results were seen after 2 hours of reperfusion. After 72 hours of reperfusion, histological damage closely correlated with calcium-calmodulin binding after variable durations of ischemia. A threshold of 10 minutes of ischemia was required to cause calcium-calmodulin binding and irreversible neuronal damage. Surviving neuronal populations showed recovery of calmodulin staining 7 days after ischemia, representing a return of free calmodulin and normal calcium homeostasis. CONCLUSIONS: These correlations between calcium-calmodulin binding, histological damage, and duration of ischemia support the causal role of calcium influx in global ischemic injury and suggest the need for very rapid intervention after ischemia if calcium-mediated damage is to be prevented.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Neuronas/patología , Animales , Encéfalo/patología , Isquemia Encefálica/patología , Umbral Diferencial , Inmunohistoquímica/métodos , Masculino , Ratas , Ratas Endogámicas , Reperfusión , Coloración y Etiquetado , Factores de TiempoRESUMEN
We evaluated several doses of cis-4-(phosphonomethyl)-2-piperidine-carboxylic acid (CGS-19755), a potent competitive N-methyl-D-aspartate (NMDA) receptor antagonist, systemically administered either before or after 20 to 30 minutes of global ischemia in rats. We measured outcome by mortality, histological damage by light microscopy, and learning ability on an eight-arm maze, and determined the drug's mechanism of action by an immunohistochemical assay of calcium-calmodulin binding. High-dose treatment begun prior to ischemia resulted in reduced cellular damage in severely ischemic hippocampal tissue, but also caused high mortality due to respiratory depression. Treatment begun 30 minutes after ischemia resulted in little histological protection but significantly improved learning ability when tested 1 month after ischemia, and did not increase mortality. Furthermore, CGS-19755, 10 mg/kg intraperitoneally, begun either before or after ischemia substantially reduced calcium influx into ischemic neurons as evidenced by reduced calcium-calmodulin binding. We conclude that CGS-19755 prevents calcium entry into ischemic neurons and may be effective therapy for very acute cerebral ischemia.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Ataque Isquémico Transitorio/tratamiento farmacológico , Ácidos Pipecólicos , Piperidinas/uso terapéutico , Receptores de Neurotransmisores/fisiología , Animales , Relación Dosis-Respuesta a Droga , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/mortalidad , Masculino , Piperidinas/farmacología , Ratas , Ratas Endogámicas , Receptores de N-Metil-D-Aspartato , Receptores de Neurotransmisores/efectos de los fármacosRESUMEN
We correlated the efficacy of several clinically relevant pharmacotherapies with their ability to prevent calcium influx into neurons and subsequent binding to calmodulin. We studied the administration of CGS 19755, nimodipine, nicardipine, and combinations of these drugs before or immediately after ischemia in globally ischemic rats. Calcium-calmodulin binding was graded by an immunohistochemical assay after 2 and 24 hours of reperfusion (n = 5-6 at each time period), and histologic damage was graded by light microscopy after 72 hours of reperfusion (n = 6). Calcium-calmodulin binding correlated with the severity of delayed histologic damage in various brain regions. In untreated ischemic control rats, marked calcium-calmodulin binding was seen in CA1 and CA3 after 24 hours of reperfusion (p less than or equal to 0.01). Administered before ischemia, CGS 19755 prevented calcium-calmodulin binding across all brain regions after 2 and 24 hours of reperfusion compared with controls (p less than or equal to 0.05). This effect was most prominent in CA3 and CA1, where the drug also reduced delayed neuronal damage (p less than or equal to 0.05). Lower doses or postischemic administration of CGS 19755, nimodipine, nicardipine, and a combination of postischemic CGS 19755 and nicardipine had a more limited effect on calcium-calmodulin binding and did not protect against delayed neuronal damage.(ABSTRACT TRUNCATED AT 250 WORDS)