Neuroprotective Dose Research Articles

Neural and Immune Consequences of Traumatic Brain Injury

Neural and Immune Consequences of Traumatic Brain Injury

A small molecule TrkB ligand reduces motor impairment and neuropathology in R6/2 and BACHD mouse models of Huntington's disease.

Loss of neurotrophic support in the striatum caused by reduced brain-derived neurotrophic factor (BDNF) levels plays a critical role in Huntington's disease (HD) pathogenesis. BDNF acts via TrkB and p75 neurotrophin receptors (NTR), and restoring its signaling is a prime target for HD therapeutics. Here we sought to determine whether a small molecule ligand, LM22A-4, specific for TrkB and without effects on p75(NTR), could alleviate HD-related pathology in R6/2 and BACHD mouse models of HD. LM22A-4 was administered to R6/2 mice once daily (5-6 d/week) from 4 to 11 weeks of age via intraperitoneal and intranasal routes simultaneously to maximize brain levels. The ligand reached levels in the R6/2 forebrain greater than the maximal neuroprotective dose in vitro and corrected deficits in activation of striatal TrkB and its key signaling intermediates AKT, PLCγ, and CREB. Ligand-induced TrkB activation was associated with a reduction in HD pathologies in the striatum including decreased DARPP-32 levels, neurite degeneration of parvalbumin-containing interneurons, inflammation, and intranuclear huntingtin aggregates. Aggregates were also reduced in the cortex. Notably, LM22A-4 prevented deficits in dendritic spine density of medium spiny neurons. Moreover, R6/2 mice given LM22A-4 demonstrated improved downward climbing and grip strength compared with those given vehicle, though these groups had comparable rotarod performances and survival times. In BACHD mice, long-term LM22A-4 treatment (6 months) produced similar ameliorative effects. These results support the hypothesis that targeted activation of TrkB inhibits HD-related degenerative mechanisms, including spine loss, and may provide a disease mechanism-directed therapy for HD and other neurodegenerative conditions.

Dose-Dependent Protective Effect of Bisperoxovanadium against Acute Cerebral Ischemia in a Rat Model of Ischemia/Reperfusion Injury

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a dual-specificity lipid and protein phosphatase. The loss of PTEN was originally discovered in numerous human cancers. PTEN inhibition by bisperoxovanadium (bpV) reduces neurological damage after ischemic brain injury. The purpose of this study was to identify the optimal neuroprotective dose of bpV when administrated after focal ischemia/reperfusion (I/R) injury in rats. Focal I/R injury was induced using the middle cerebral artery occlusion method. bpV at doses of 0.25, 0.50 and 1.0 mg/kg were injected intraperitoneally just after reperfusion, with saline serving as a vehicle control. A maximal reduction in brain injury was observed with 1.0 mg/kg bpV. This dose of bpV also significantly blocked apoptosis in the penumbral cortex of rats. This beneficial effect was associated with the increasing levels of Akt phosphorylation in the penumbral cortex. These results demonstrate that the pharmacological inhibition of PTEN protects against I/R injury in a dose-dependent manner and the protective effect might be induced through upregulation of the phosphoinositide-3 kinase/Akt pro-survival pathway, suggesting a new therapeutic strategy to combat ischemic brain injury.

Plasma topiramate concentrations resulting from doses associated with neuroprotection against white matter injury and stroke in two strains of rat pups

Cerebral white matter (WM) injury and stroke are common neuropathological injuries in newborns with congenital heart defects (CHDs) requiring surgery. Previous investigations in Long Evans rat pups subjected to hypoxia-ischemia found that intraperitoneal (i.p.) topiramate (TPM) at 30 mg/kg, but not 50 mg/kg, conferred neuroprotection. In Sprague-Dawley pups, a dose of 30 mg/kg protected against stroke. Concentrations associated with neuroprotective doses were not measured. The aims of this investigation were to determine concentrations associated with neuroprotective doses and to investigate the pharmacokinetics (PK) of i.p. TPM. Concentration-time data following administration of 30 and 50 mg/kg doses were analyzed using nonlinear mixed-effect modeling. Mean predicted steady-state maximum and average concentrations following 30 mg/kg TPM were 31.3 and 16.8 μg/ml in Long Evans and 39.9 and 24.4 μg/ml in Sprague-Dawley pups. Mean predicted steady-state maximum and average concentrations following 50 mg/kg TPM were 52.1 and 28.1 μg/ml in Long Evans and 66.5 and 40.6 μg/ml in Sprague-Dawley pups. The apparent clearance (CL/F) and apparent volume of distribution (V/F) were 0.0470 ml/min and 22.2 ml, respectively, for Long Evans and 0.0325 ml/min and 19.7 ml, respectively, for Sprague-Dawley pups. TPM concentrations associated with neuroprotective doses were determined. Body size and strain were significant covariates on CL/F and V/F. Results provide targets for future neuroprotection studies.

The Therapeutic Profile of Rolipram, PDE Target and Mechanism of Action as a Neuroprotectant following Spinal Cord Injury

The extent of damage following spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of cyclic adenosine monophosphate (cyclic AMP), via administration of the phosphodiesterase 4 (PDE4) inhibitor Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following contusive SCI in rat as well as its prominent PDE target and putative mechanism of protection. Rolipram or vehicle control (10% ethanol) was given subcutaneously (s.c.) daily for 2 wk post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (given at 1 h PI) increased neuronal survival; 0.5 mg to 1.0 mg/kg protected oligodendrocytes and 1.0 mg/kg produced optimal preservation of central myelinated axons. Ethanol also demonstrated significant neuronal and oligo-protection; though the preservation provided was significantly less than Rolipram. Subsequent use of this optimal Rolipram dose, 1.0 mg/kg, via different routes (i.v., s.c. or oral, 1 h PI), demonstrated that i.v. administration produced the most significant and consistent cyto- and axo- protection, although all routes were effective. Examination of the therapeutic window for i.v. Rolipram (1.0 mg/kg), when initiated between 1 and 48 h after SCI, revealed maximal neuroprotection at 2 h post-SCI, although the protective efficacy of Rolipram could still be observed when administration was delayed for up to 48 h PI. Importantly, use of the optimal Rolipram regimen significantly improved locomotor function after SCI as measured by the BBB score. Lastly we show SCI-induced changes in PDE4A, B and D expression and phosphorylation as well as cytokine expression and immune cell infiltration. We demonstrate that Rolipram abrogates SCI-induced PDE4B1 and PDE4A5 production, PDE4A5 phosphorylation, MCP-1 expression and immune cell infiltration, while preventing post-injury reductions in IL-10. This work supports the use of Rolipram as an acute neuroprotectant following SCI and defines an optimal administration protocol and target for its therapeutic application.

Neuroprotective effect of human placental extract on hypoxic–ischemic brain injury in neonatal rats

We investigated the neuroprotective effects of human placental extracts (HPE) and the effects of HPE on recovery of cognitive and behavioral function on hypoxic–ischemic brain injury in the newborn rat. The right common carotid arteries of 7-day-old rats were coagulated, and rats were then exposed to 8% oxygen. Immediately before and again at three times after the hypoxia–ischemia (pre-treatment group), and immediately after and three times again after hypoxia–ischemia (post-treatment group), the rats were intraperitoneally injected with HPE (0.1, 0.25, or 0.5mL/10g/dose). No-treatment rats received saline only. On postnatal day 12, brains were removed and gross morphological damage was evaluated. To quantify the severity of brain injury, bilateral cross-sectional areas of the anterior commissural and posterior hippocampal levels were analyzed with NIH Image. Assessments of the open field activity levels at 2, 4, 6 and 8week and, the Morris water maze test at 8weeks after hypoxia–ischemia were carried out according to standard methods. HPE pre-treatment decreased the incidence of liquefactive cerebral infarction, at an optimally neuroprotective dose of 0.5mL/10g/dose (P<0.05). In the Morris water maze test, the group injected with HPE at 0.5mL/10g/dose concentration showed shorter escape latencies than the no-treatment group (P<0.05). These findings support a protective effect of the HPE treatment on neuronal integrity and cognitive function following hypoxic–ischemic brain injury. Injected at an appropriate dose prior to exposure, HPE may significantly reduce or prevent hypoxic–ischemic injury in the immature brain.

The clinically available NMDA receptor antagonist, memantine, exhibits relative safety in the developing rat brain

The N-methyl- d-aspartate glutamate receptor (NMDAR) has been implicated in preterm brain injury (periventricular leukomalacia (PVL)) and represents a potential therapeutic target. However, the antagonist dizocilpine (MK-801) has been reported to increase constitutive neuronal apoptosis in the developing rat brain, limiting its clinical use in the developing brain. Memantine is another use-dependent NMDAR antagonist with shorter binding kinetics and has been demonstrated to be protective in a rat model of PVL, without effects on normal myelination or cortical growth. To further evaluate the safety of memantine in the developing brain, we demonstrate here that, in contrast to MK-801, memantine at neuroprotective doses does not increase neuronal constitutive apoptosis. In addition, there are no long-term alterations in the expression of NMDAR subunits, AMPAR subunits, and two markers of synaptogenesis, Synapsin-1 and PSD95. Evaluating clinically approved drugs in preclinical neonatal animal models of early brain development is an important prerequisite to considering them for clinical trial in preterm infants and early childhood.

Psychopharmacological Neuroprotection in Neurodegenerative Disease: Assessing the Preclinical Data

Psychopharmacological Neuroprotection in Neurodegenerative Disease: Assessing the Preclinical Data

Psychopharmacological Neuroprotection in Neurodegenerative Disease: Assessing the Preclinical Data

This manuscript reviews the preclinical in vitro, ex vivo, and nonhuman in vivo effects of psychopharmacological agents in clinical use on cell physiology with a view toward identifying agents with neuroprotective properties in neurodegenerative disease. These agents are routinely used in the symptomatic treatment of neurodegenerative disease. Each agent is reviewed in terms of its effects on pathogenic proteins, proteasomal function, mitochondrial viability, mitochondrial function and metabolism, mitochondrial permeability transition pore development, cellular viability, and apoptosis. Effects on the metabolism of the neurodegenerative disease pathogenic proteins alpha-synuclein, beta-amyloid, and tau, including tau phosphorylation, are particularly addressed, with application to Alzheimer's and Parkinson's diseases. Limitations of the current data are detailed and predictive criteria for translational clinical neuroprotection are proposed and discussed. Drugs that warrant further study for neuroprotection in neurodegenerative disease include pramipexole, thioridazine, risperidone, olanzapine, quetiapine, lithium, valproate, desipramine, maprotiline, fluoxetine, buspirone, clonazepam, diphenhydramine, and melatonin. Those with multiple neuroprotective mechanisms include pramipexole, thioridazine, olanzapine, quetiapine, lithium, valproate, desipramine, maprotiline, clonazepam, and melatonin. Those best viewed circumspectly in neurodegenerative disease until clinical disease course outcomes data become available, include several antipsychotics, lithium, oxcarbazepine, valproate, several tricyclic antidepressants, certain SSRIs, diazepam, and possibly diphenhydramine. A search for clinical studies of neuroprotection revealed only a single study demonstrating putatively positive results for ropinirole. An agenda for research on potentially neuroprotective agent is provided.

Intrathecal Corticoids in Permanent Focal Cerebral Ischemia in Rats. Part I: A New Therapeutic Approach in the Acute Phase

Intrathecally, triamcinolone acetonide (TCA) was suggested to have neuroprotective efficacy on infarction volume in acute focal cerebral ischemia in rats. In the first dose-finding study, TCA in five different doses or saline was administered into the cisterna magna of 12 rats, each 30 mins after endovascular occlusion of the middle cerebral artery (MCAO). In the second magnet resonance controlled confirmation study, the most neuroprotective dose was compared with controls in each of the 15 rats. Infarction volume was calculated at 24 h by 2.3.5 triphenyl-tetrazolium-chloride staining. Compared with controls (18.2%), infarction volume was significantly reduced using TCA at a dose of 0.012 mg/kg body weight (BW) (13.4%, P=0.04). TCA at doses of 0.03 (17.7%, P=0.84), 0.006 (15.9%, P=0.24), and 0.003 mg/kg BW (14.5%, P=0.11) did not significantly reduce infarction size. TCA 0.3 mg/kg BW resulted in bilateral infarction with increased infarction volume (19.8%, P=0.49). Magnetic resonance imaging confirmed successful MCAO and intrathecal administration. In experiment 2 compared with controls (20.0%), infarction volume was significantly reduced using TCA 0.012 mg/kg (13.4%, P=0.02). Intrathecally, TCA may significantly reduce infarction volume in acute focal cerebral ischemia in rats. Further studies are necessary to define the value of this therapy.

Subchronic Alpha-Linolenic Acid Treatment Enhances Brain Plasticity and Exerts an Antidepressant Effect: A Versatile Potential Therapy for Stroke

Omega-3 polyunsaturated fatty acids are known to have therapeutic potential in several neurological and psychiatric disorders. However, the molecular mechanisms of action underlying these effects are not well elucidated. We previously showed that alpha-linolenic acid (ALA) reduced ischemic brain damage after a single treatment. To follow-up this finding, we investigated whether subchronic ALA treatment promoted neuronal plasticity. Three sequential injections with a neuroprotective dose of ALA increased neurogenesis and expression of key proteins involved in synaptic functions, namely, synaptophysin-1, VAMP-2, and SNAP-25, as well as proteins supporting glutamatergic neurotransmission, namely, V-GLUT1 and V-GLUT2. These effects were correlated with an increase in brain-derived neurotrophic factor (BDNF) protein levels, both in vitro using neural stem cells and hippocampal cultures and in vivo, after subchronic ALA treatment. Given that BDNF has antidepressant activity, this led us to test whether subchronic ALA treatment could produce antidepressant-like behavior. ALA-treated mice had significantly reduced measures of depressive-like behavior compared with vehicle-treated animals, suggesting another aspect of ALA treatment that could stimulate functional stroke recovery by potentially combining acute neuroprotection with long-term repair/compensatory plasticity. Indeed, three sequential injections of ALA enhanced protection, either as a pretreatment, wherein it reduced post-ischemic infarct volume 24 h after a 1-hour occlusion of the middle cerebral artery or as post-treatment therapy, wherein it augmented animal survival rates by threefold 10 days after ischemia.

Inhibiting pro-death NMDA receptor signaling dependent on the NR2 PDZ ligand may not affect synaptic function or synaptic NMDA receptor signaling to gene expression

NMDA receptors (NMDARs) mediate ischemic brain damage, in part through interactions of the PDZ ligand of NR2 subunits with the PDZ domain proteins PSD-95 and neuronal nitric oxide synthase located within the NMDAR signaling complex. We have recently shown that this PDZ ligand-dependent pathway promotes neuronal death via p38 activation. A peptide mimetic of the NR2B PDZ ligand (TAT-NR2B9c) reduces p38-mediated death in vitro and p38-dependent ischemic damage in vivo. In the absence of the PDZ ligand-p38 pathway, such as in TAT-NR2B9c-treated neurons, or in NMDAR-expressing non-neuronal cells, NMDAR-dependent excitotoxicity is mediated largely by JNK and requires greater Ca2+ influx. A major reason for blocking pro-death signaling events downstream of the NMDAR as an anti-excitotoxic strategy is that it may spare physiological synaptic function and signaling. We find that neuroprotective doses of TAT-NR2B9c do not alter the frequency of spontaneous synaptic events within networks of cultured cortical neurons nor is mini-EPSC frequency altered. Furthermore, TAT-NR2B9c does not inhibit the capacity of synaptic NMDAR activity to promote neuroprotective changes in gene expression, including the up-regulation of PACAP via CREB, and suppression of the pro-oxidative FOXO target gene Txnip. Thus, while the NR2 PDZ ligand does not account for all the excitotoxic effects of excessive NMDAR activity, these findings underline the value of the specific targeting of death pathways downstream of the NMDAR.

P1-014: At therapeutic, neuroprotective doses, memantine blocks NMDA receptors and does not affect learning in nonimpaired rats

P1-014: At therapeutic, neuroprotective doses, memantine blocks NMDA receptors and does not affect learning in nonimpaired rats

RGD domains neuroprotect the immature brain by a glial‐dependent mechanism

Integrin binding to extracellular matrix ligands, including those presenting RGD motifs, modulate diverse cellular processes. In the brain, many endogenous RGD-containing molecules are induced after damage. Previously, the gene therapy vector termed NLSCt, which displays an RGD motif, was shown to neuroprotect after immature brain excitotoxicity. We analyze whether neuroprotection is mediated by the RGD motif. RGD-containing synthetic peptide GPenGRGDSPCA (GPen) was injected 2 hours after N-methyl-D-aspartate-mediated excitotoxicity to the postnatal day 9 rat brain. Damage and glial/inflammatory response were evaluated 3 days later. In addition, the neuroprotective effect of GPen and NLSCt after N-methyl-D-aspartate-induced cell death was also analyzed in vitro using neuron-purified and mixed neuron-glia primary cultures. To further characterize whether the neuroprotective effect was mediated by glial-derived soluble factors, we also tested the protective ability of conditioned media from RGD-treated microglia, astrocyte, or mixed glia cultures. Animals treated with GPen peptide showed functional improvement, a significant reduction in lesion volume up to 28%, and a decrease in the number of degenerating neurons. In addition, N-methyl-D-aspartate-injected animals treated with both RGD-containing molecules at the neuroprotective doses showed a significant increase in microglial reactivity and microglia/macrophage cell number, but no differences in neutrophil infiltration and the astroglial response. Finally, in vitro studies showed that the neuroprotective effect was observed in mixed neuron-glia, but not in neuron-purified cultures. Conditioned media from RGD-treated microglial, astroglial, and mixed-glial cultures were not protective. These results suggest that RGD-containing molecules neuroprotect by a glial-dependent mechanism.

NMDA Receptors, Glial Cells, and Clinical Medicine

Recent reports have overturned a series of dogmas that have been well entrenched in the neuroscience literature concerning NMDA-type glutamate receptors (NMDARs). The new data show that NMDARs exist on the myelin sheath formed by oligodendrocytes, that an uncompetitive NMDAR antagonist has successfully passed human clinical trials, and that NMDARs trigger multiple deleterious cascades to inflict cellular damage on both neurons and glia during cerebral ischemia (stroke). These recent findings bode well for clinical intervention with NMDAR antagonists in more neurological disorders than previously thought, including multiple sclerosis, cerebral palsy (periventricular leukomalacia), and spinal cord injury.

Therapeutic Time Window and Dose Dependence of Neuroprotective Effects of Sodium Orthovanadate following Transient Middle Cerebral Artery Occlusion in Rats

Vanadium is widely distributed in the environment and exhibits various biological and physiological effects in the human body. We previously documented the neuroprotective effect of sodium orthovanadate (SOV) against in rodents i.v. injected with 2 ml/kg 50 mM SOV just after the induction of middle cerebral artery occlusion (MCAO; 0 min post-MCAO). To evaluate its potential clinical use, we determined here therapeutic time window (0, 45, and 90 min post-MCAO) and the neuroprotective dose (2 ml/kg, 12.5, 25, 37.5, and 50 mM) of SOV in rats. A single injection of 50 mM SOV at 0 or 45 min post-MCAO produced similar neuroprotective effects, and even 50 mM delivered 90 min post-MCAO exerted significant neuroprotection. Although the maximal neuroprotective effect was obtained at 50 mM SOV, 25 mM injected once and 12.5 mM delivered at 0 and 45 min post-MCAO significantly reduced the infarct volume. We also documented that SOV treatment ameliorates ischemic neuronal cell injury via the activation of both protein kinase B (Akt) and extracellular signal-regulated kinase (ERK), inhibits serum glucose, and elicits the gradual recovery of regional cerebral blood flow (rCBF) after transient MCAO in rats. To elucidate the important factor(s) involved in the neuronal protection afforded by SOV, we measured Akt and ERK activity, physiological parameters, blood glucose levels, and rCBF following various SOV treatments. In conclusion, Akt activation was the most important factor in SOV-induced neuroprotection; ERK activation, the gradual recovery of rCBF, and decreased blood glucose were weak contributors.

The pre-ischaemic neuroprotective effect of a novel polyamine antagonist, N1-dansyl-spermine in a permanent focal cerebral ischaemia model in mice

The polyamine sites on the NMDA receptor complex offer a therapeutic target for focal ischaemia, potentially devoid of most side effects associated with NMDA antagonists. In this study, we investigated the effect of a novel polyamine antagonist, N 1-dansyl-spermine (0.5–10 mg kg −1) in a permanent focal cerebral ischaemia model in mice, and compared its effect to that of MK-801 (0.3–3 mg kg −1) following administration 30 min prior to ischaemia. A battery of histological and behavioural tests was employed following permanent middle cerebral artery occlusion to assess any neuroprotective effect. Following middle cerebral artery occlusion, N 1-dansyl-spermine (1–5 mg kg −1) and MK-801 (1 or 3 mg kg −1) caused a comparable and significant reduction in the percentage hemisphere lesion volume. Similarly, both drugs significantly reduced oedema and neurological deficit score to a similar extent. Locomotor activity in MCAO mice was not significantly improved by MK-801 or N 1-dansyl-spermine, although N 1-dansyl-spermine induced a trend towards significant improvement. Significant improvement in rotarod performance was observed at neuroprotective doses with both drugs. Upon comparison of the profile of effects, N 1-dansyl-spermine at least matched the effectiveness of MK-801 as a neuroprotective agent in this model. In addition, in sham-operated control mice, N 1-dansyl-spermine was well tolerated, in contrast to the pronounced adverse effects of MK-801 on locomotor activity and rotarod performance. In conclusion, this study has shown that N 1-dansyl-spermine is as effective a neuroprotective drug as MK-801 in this model. Moreover, in contrast to MK-801, N 1-dansyl-spermine could be a promising therapeutic candidate for stroke as it is well tolerated at neuroprotective doses in sham-operated animals.

Time-dependent changes in CRH concentrations and release in discrete brain regions following global ischemia: effects of MK-801 pretreatment

The excitatory actions of corticotropin-releasing hormone (CRH) in the brain and the neuroprotective effects of CRH antagonists in models of ischemia suggest a role for this peptide in the cascade of events leading to cellular damage. The present study aimed to characterize endogenous activation of CRH in discrete brain regions following global ischemia. Time-dependent changes in CRH concentrations were assessed in 10 brain regions including hippocampal, parahippocampal, and hypothalamic regions as well as the amygdala and the frontal cortex at three post-ischemic intervals: 4, 24, and 72 h (Experiment 1). The impact of pretreatment with a neuroprotective dose of the NMDA antagonist (5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; hydrogen maleate) on 24-h ischemia-induced CRH concentrations in the 10 brain regions was also determined (Experiment 2). In vivo microdialysis was used to assess dynamic fluctuations in CRH release at the dorsal hippocampus (CA1 pyramidal layer) and central nucleus of the amygdala (CeA; Experiment 3). Our findings revealed a rapid elevation of CRH concentrations at the piriform cortex (Pir) and hypothalamic nuclei following global ischemia. This was followed by decreased CRH concentrations at the amygdala, the frontal cortex (FC), the CA3, and the hypothalamus 24-h post-ischemia. MK-801 reversed the decreases in the hypothalamic nuclei but not in the other brain regions. Seventy-two hours post-ischemia, CRH levels returned to control values in all regions except the dentate gyrus (DG) where elevated CRH levels were observed. In vivo, a significant increase in CRH release in response to global ischemia was found at the CeA with no alterations at the CA1. These findings support brain region-specific ischemia-induced CRH alterations and suggest that CRH actions to mediate neuronal damage at the hippocampal CA1 layer may be indirect.

Molecular mechanisms of neuroprotective action of immunosuppressants--facts and hypotheses.

Cyclosporin A (CsA) and FK506 (Tacrolimus) are short polypeptides which block the activation of lymphocytes and other immune system cells. Immunosuppressants exert neuroprotective and neurotrophic action in traumatic brain injury, sciatic nerve injury, focal and global ischemia in animals. Their neuroprotective actions are not understood and many hypotheses have been formed to explain such effects. We discuss a role of drug target--calcineurin in neuroprotective action of immunosuppressants. Protein dephosphorylation by calcineurin plays an important role in neuronal signal transduction due to its ability to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In vitro FK506 protects cortex neurons from NMDA-induced death, augments NOS phosphorylation inhibiting its activity and NO synthesis. However, in vivo experiments demonstrated that FK506 in neuroprotective doses did not block excitotoxic cell death nor did it alter NO production during ischemia/reperfusion. Tissue damage in ischemia is the result of a complex pathophysiological cascade, which comprises a variety of distinct pathological events. Resident non-neuronal brain cells respond rapidly to neuronal cell death and may have both deleterious and useful role in neuronal damage. There is increasing evidence that reactive gliosis and post-ischemic inflammation involving microglia contribute to ischemic damage. We have demonstrated that FK506 modulates hypertrophic/proliferative responses and proinflammatory cytokine expression in astrocytes and microglia in vitro and in focal transient brain ischemia. Our findings suggest that astrocytes and microglia are direct targets of FK506 and modulation of glial response and inflammation is a possible mechanism of FK506-mediated neuroprotection in ischemia.

Reduced progesterone metabolites protect rat hippocampal neurones from kainic acid excitotoxicity in vivo.

The ovarian hormone progesterone is neuroprotective in some animal models of neurodegeneration. Progesterone actions in the brain may partly be mediated by the locally produced metabolites 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone. The neuroprotective effects of these two metabolites of progesterone were assessed in this study. Ovariectomized Wistar rats were injected with kainic acid, to induce excitotoxic neuronal death in the hippocampus, and with different doses of 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone. The number of surviving neurones in the hilus of the dentate gyrus of the hippocampus was assessed with the optical disector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurones and in the induction of vimentin expression in reactive astrocytes, a sign of neural damage. Low doses of 5alpha-dihydroprogesterone (0.25 and 0.5 mg/kg body weight, b.w.) prevented the loss of hilar neurones and the appearance of vimentin immunoreactivity in astrocytes. Higher doses (1-2 mg/kg b.w.) were not neuroprotective. By contrast, low doses of 3alpha,5alpha-tetrahydroprogesterone (0.25-1 mg/kg b.w.) were unable to protect the hilus from kainic acid while higher doses (2-4 mg/kg b.w.) were protective. The different optimal neuroprotective doses of 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone suggest that these two steroids may protect neurones using different mechanisms. The neuroprotective effects of 3alpha,5alpha-tetrahydroprogesterone may be exerted by the inhibition of neuronal activity via the GABAA receptor. This latter possibility is supported by the observation that 3beta,5alpha-tetrahydroprogesterone, an isomer of 3alpha,5alpha-tetrahydroprogesterone that does not bind to GABAA receptor, was not neuroprotective. In summary, our findings suggest that progesterone neuroprotective effects may be, at least in part, mediated by its reduced metabolites 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone.