Synthetic structural modifications of neurosteroid pregnanolone sulfate: Assessment of neuroprotective effects in vivo.

Neuroactive steroid 20-oxo-5ß-pregnan-3α-yl L-glutamyl 1-ester (PA-Glu), a synthetic analogue of naturally occurring 20-oxo-5ß-pregnan-3α-yl sulfate (pregnanolone sulfate, PA-S), inhibits N-methyl-D-aspartate (NMDA) receptors and possesses neuroprotective properties and minimal adverse effects. Herein, we report in vivo effects of new structural modifications of the PA-S molecule: a nonpolar modification of the steroid D-ring (5ß-androstan-3α-yl L-glutamyl 1-ester, AND-Glu), attachment of a positively charged group to C3 (20-oxo-5ß-pregnan-3α-yl L-argininate dihydrochloride salt, PA-Arg) and their combination (5ß-androstan-3α-yl L-argininate dihydrochloride salt, AND-Arg). The first aim of this study was to determine the structure-activity relationship for neuroprotective effects in a model of excitotoxic hippocampal damage in rats, based on its behavioral correlate in Carousel maze. The second aim was to explore side effects of neuroprotective steroids on motor functions, anxiety (elevated plus maze) and locomotor activity (open field) and the effect of their high doses in mice. The neuroprotective properties of PA-Glu and AND-Glu were proven, with the effect of the latter appearing to be more pronounced. In contrast, neuroprotective efficacy failed when positively charged molecules (PA-Arg, AND-Arg) were used. AND-Glu and PA-Glu at the neuroprotective dose (1 mg/kg) did not unfavorably influence motor functions of intact mice. Moreover, anxiolytic effects of AND-Glu and PA-Glu were ascertained. These findings corroborate the value of research of steroidal inhibitors of NMDA receptors as potential neuroprotectants with slight anxiolytic effect and devoid of behavioral adverse effects. Taken together, the results suggest the benefit of the nonpolar D-ring modification, but not of the attachment of a positively charged group to C3.

Concurrent assessment of memory for object and place: Evidence for different preferential importance of perirhinal cortex and hippocampus and for promnestic effect of a neurokinin-3 R agonist.

We here explore the utility of a paradigm that allows the simultaneous assessment of memory for object (what) and object location (where) and their comparative predominance. Two identical objects are presented during a familiarity trial; during the test trial one of these is displaced, and a new object is presented in a familiar location. When tested 5 or 80min later, rats explored both the novel and the displaced objects more than two familiar stationary objects, indicating intact memory for both, object and place. When tested 24h later rats explored the novel object more than the displaced familiar one, suggesting that forgetting differently influenced object and place memory, with memory for object being more robust than memory for place. Animals that received post-trial administration of the neurokinin-3 receptor agonist senktide and were tested 24h later, now explored the novel and displaced objects equally, suggesting that the treatment prevented the selective decay of memory for location. Next, animals received NMDA lesions in either the perirhinal cortex or the hippocampus, which are hypothesized to be preferentially involved in memory for objects and memory for place, respectively. When tested 5 or 80min later, the perirhinal cortex lesion group explored the displaced object more, indicating relatively deficient object memory, while the hippocampal lesion led to the opposite pattern, demonstrating comparatively deficient place memory. These results suggest different preferential engagement of the perirhinal cortex and hippocampus in their processing of memory for object and place. This preference test lends itself to application in the comparison of selective lesions of neural sites and projection systems as well as to the assessment of possible preferential action of pharmacological agents on neurochemical processes that subserve object vs place learning.

Toxicity assessment of intravitreal triamcinolone and bevacizumab in a retinal explant mouse model using two-photon microscopy.

PURPOSE: Intravitreal drug administration leads to high intraocular concentrations with potentially toxic effects on ocular tissues. This study was an assessment of the toxicity of triamcinolone and bevacizumab in living retinal explants using two-photon (2P) microscopy. METHODS: Wild-type mice received intravitreal injections of triamcinolone, bevacizumab, or vehicle. Ten and 45 days after injection, wholemounted retinal explants were incubated with the fluorescent dye sulforhodamine 101 (SR101) to analyze morphology and tissue damage with 2P microscopy ex vivo. Retinas that received the same treatment were stained for apoptosis (TUNEL) and glial activation (GFAP). An intravitreal injection of NMDA (N-methyl-d-aspartate) was used as a positive control to ensure the fidelity of detection of retinal damage with ex vivo 2P microscopy. RESULTS: Overall retinal morphology was undisturbed after all procedures and time points. NMDA injection resulted in a strong increase in the number of SR101-labeled cells and increased apoptosis and glial activation when compared with sham-injected eyes. This result was in contrast to exposure to bevacizumab, which caused no appreciable damage. After triamcinolone treatment, marked damage in the inner retina was observed. However, damaged cells were restricted to sharply demarcated areas, and only mild changes in TUNEL-positive cells and GFAP activation was observed when compared to sham-injected eyes. CONCLUSIONS: 2P microscopy in combination with SR101 staining allows fast morphologic assessment of living retinal explants and can be used to evaluate adverse effects on retinal viability of test substances. Bevacizumab treatment did not cause any detectable retinal damage, whereas triamcinolone was associated with substantial, although spatially restricted, damage.

Assessment of primary neuronal culture as a model for soman-induced neurotoxicity and effectiveness of memantine as a neuroprotective drug.

An in vitro mammalian model neuronal system to evaluate the intrinsic toxicity of soman and other neurotoxicants as well as the efficacy of potential countermeasures was investigated. The link between soman toxicity, glutamate hyperactivity and neuronal death in the central nervous system was investigated in primary dissociated cell cultures from rat hippocampus and cerebral neocortex. Exposure of cortical or hippocampal neurons to glutamate for 30 min produced neuronal death in almost 80% of the cells examined at 24 h. Hippocampal neurons exposed to soman for 15-120 min at 0.1 microM concentration caused almost complete inhibition (> or = 90%) of acetylcholinesterase but failed to show any evidence of effects on cell viability, indicating a lack of direct cytotoxicity by this agent. Acetylcholine (ACh, 0.1 mM), alone or in combination with soman, did not potentiate glutamate toxicity in hippocampal neurons. Memantine, a drug used for the therapy of Parkinson's disease, spasticity and other brain disorders, significantly protected hippocampal and cortical neurons in culture against glutamate and N-methyl-D-aspartate (NMDA) excitotoxicity. In rats a single dose of memantine (18 mg/kg) administered 1 h prior to a s.c. injection of a 0.9 LD50 dose of soman reduced the severity of convulsions and increased survival. Survival, however, was accompanied by neuronal loss in the frontal cortex, piriform cortex and hippocampus.