Highly efficient small interfering RNA delivery to primary mammalian neurons induces MicroRNA-like effects before mRNA degradation.

The study of protein function in neurons has been hindered by the lack of highly efficient, nontoxic methods of inducing RNA interference in such cells. Here we show that application of synthetic small interfering RNA (siRNA) linked to the vector peptide Penetratin1 results in rapid, highly efficient uptake of siRNA by entire populations of cultured primary mammalian hippocampal and sympathetic neurons. This treatment leads to specific knock-down of targeted proteins within hours without the toxicity associated with transfection. In contrast to current methods, our technique permits study of protein function across entire populations with minimal disturbance of complex cellular networks. Using this technique, we found that protein knock-down (evident after 6 hr) precedes any decrease in targeted message (evident after 24 hr), suggesting an early, translational repression by perfectly targeted siRNAs.

Caspase function in neuronal death: delineation of the role of caspases in ischemia.

Cerebral ischemia is one of the major causes of morbidity and mortality in the Western world. Despite extensive research, adequate therapies are still elusive. Neuronal degeneration and death are hallmarks of stroke/ischemia. Understanding how the death machinery executes neuronal death in ischemia will provide therapeutic targets. Key to the death machinery are caspases: the family of cell death proteases. While much data has been published regarding caspase involvement in models of ischemia, the pathways have not been thoroughly defined. The specification of the caspases critical for death has been hampered by the use of non-specific reagents. Thus many conclusions about specificity are unwarranted. In this review we discuss how caspases can be measured and review the existing knowledge of the roles of specific caspases in ischemia. We also discuss approaches to determining the molecules that execute ischemic death.

Delayed cell death related to acute cerebral blood flow changes following subarachnoid hemorrhage in the rat brain.

OBJECT: The authors tested the hypotheses that subarachnoid hemorrhage (SAH) leads to delayed cell death with the participation of apoptotic-like mechanisms and is influenced by the degree of acute decrease in the cerebral blood flow (CBF) following hemorrhage. METHODS: Subarachnoid hemorrhage was induced in rats by endovascular perforation of the internal carotid artery or injection of blood into the prechiasmatic cistern. Cerebral blood flow was measured using laser Doppler flowmetry for 60 minutes. Brain sections stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) showed DNA fragmentation at 2 and 7 days after both methods of inducing SAH in one third to two thirds of the surviving animals in the different experimental groups. More than 80% of the TUNEL-positive cells were neuron-specific nuclear protein-positive (neurons), but immunoreactivity to glial fibrillary acidic protein (astrocytes) and transferrin (oligodendrocytes) were markedly decreased in TUNEL-positive areas. Most of the TUNEL-positive cells displayed chromatin condensation and/or blebs and immunostained for increased Bax; approximately 50% of them were immunoreactive to cleaved caspase-3 and a few to Bcl-2. The duration of the acute CBF decrease below 30% of the baseline level was related to the degree of TUNEL staining. CONCLUSIONS: Subarachnoid hemorrhage resulted in delayed cell death in a large proportion, but not all, of the surviving animals. The acute CBF decrease was related to the degree of subsequent cell death. These findings indicated the relevance of apoptotic-like pathways. There appears to be a temporal therapeutic window during which adequate treatment might reduce the final damage following SAH.

A new experimental model in rats for study of the pathophysiology of subarachnoid hemorrhage.

A new experimental model of subarachnoid hemorrhage (SAH) in rats is described. A needle was stereotaxically placed in the prechiasmatic cistern and 300, 250 or 200 microl of blood was injected manually, keeping the intracranial pressure (ICP) at the mean arterial blood pressure (MABP) level. An acceptable mortality was observed only after injection of 200 microl of blood. In this group, MABP and ICP increased immediately after SAH, but soon approached baseline levels. The subarachnoid blood was mainly distributed in the basal cisternal system and its estimated volume was about 95% of the amount injected. This new model resembles clinical SAH, is very reproducible, easy to use and seems to be a suitable model for studies of the pathophysiology of SAH.

Inflammation in the brain after experimental subarachnoid hemorrhage.

OBJECTIVE: To study the occurrence of an inflammatory response in the brain after subarachnoid hemorrhage and its relation to the decrease in acute cerebral blood flow, subarachnoid blood proximity, and cell damage. METHODS: Subarachnoid hemorrhage was induced in rats via endovascular perforation of the internal carotid artery or injection of blood into the prechiasmatic cistern. Cerebral blood flow was measured by laser Doppler flowmetry for 60 minutes. After 2 and 7 days, the brains were analyzed by immunohistochemistry using the following antibodies: OX6, ED1, intercellular adhesion molecule 1, tumor necrosis factor alpha, interleukin-1beta, interleukin-6, inducible nitric oxide synthase, and nestin. Deoxyribonucleic acid fragmentation was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling. RESULTS: In approximately half of the surviving animals (0-92%, depending on the marker and subgroup), signs of inflammation were detected. The most common findings were increased immunoreactivity to nestin, ED1, OX6, intercellular adhesion molecule 1, and tumor necrosis factor alpha. There was great variability in the intensity and the location of the inflammatory reaction among the animals, but tissues in proximity to the extravasated blood seemed to be especially affected. A significant correlation between the duration of cerebral blood flow under 30% of the baseline and the degree of the inflammation was observed. There was a strong correspondence between areas showing deoxyribonucleic acid fragmentation and inflammation. CONCLUSION: Subarachnoid hemorrhage triggered an inflammatory reaction in the brain in a large fraction of the surviving animals, which may have contributed to cell death. Acute ischemic episodes and direct effect of blood seemed to be significant factors in its genesis.

Balancing neuronal death.

Although it is apparent that neuronal death must be tightly regulated to ensure the proper development and mature functions of the nervous system, the molecular details of this regulation are not fully understood. In multiple neurodegenerative diseases, there is inappropriate death of cells in the nervous system. A better understanding of how death is regulated in the normal nervous system can provide a framework for determining how this regulation can go awry during neurodegenerative disease. The key executioners of neuronal apoptosis, the caspases, are regulated at several levels. The endogenous inhibitor of apoptosis family of proteins, the IAPs, can suppress caspase activity. In this Mini-Review, we examine what is known about the function of IAPs in normal neuronal function and in disease.