Post-stroke infections exacerbate ischemic brain injury in middle-aged rats: immunomodulation and neuroprotection by progesterone.

We investigated the effect of delayed, prolonged systemic inflammation on stroke outcomes and progesterone (P4) neuroprotection in middle-aged rats. After transient middle cerebral artery occlusion/reperfusion (MCAO) surgery, rats received P4 (8 or 16 mg/kg) or vehicle injections at 2h, 6h and every 24h until day 7 post-occlusion. At 24h post-injury systemic inflammation was induced by giving three doses of lipopolysaccharide (LPS; 50 µg/kg, at 4h intervals) to model post-stroke infections. We measured serum brain-derived neurotrophic factor (BDNF), pro-inflammatory cytokines, and behavioral parameters at multiple times. Serum BDNF levels decreased more in the vehicle+LPS group compared to vehicle-alone at 3 and 7 days post-injury (P<0.05). Vehicle-alone showed a significant increase in interleukin-1ß, interleukin-6, and tumor necrosis factor alpha levels at different times following stroke and these levels were further elevated in the vehicle+LPS group. P4 at both doses produced a significant (P<0.05) decline in cytokine levels compared to vehicle and vehicle+LPS. P4 restored BDNF levels at 3 and 7 days post-stroke (P<0.05). Behavioral assessment (rotarod, grip strength, sensory neglect and locomotor activity tests) at 3, 5 and 7 days post-stroke revealed that the vehicle group had significant (P<0.05) deficits in all tests compared to intact controls, and performance was worse in the vehicle+LPS group. P4 at both doses produced significant functional improvement on all tests. Systemic inflammation did not show an additive effect on infarct volume but P4 at both doses showed significant infarct reduction. We suggest that post-stroke infection exacerbates stroke outcomes and P4 exerts neuroprotective/modulatory effects through its systemic anti-inflammatory and BDNF regulatory actions.

Distribution of membrane progesterone receptor alpha in the male mouse and rat brain and its regulation after traumatic brain injury.

Progesterone has been shown to exert pleiotropic actions in the brain of both male and females. In particular, after traumatic brain injury (TBI), progesterone has important neuroprotective effects. In addition to intracellular progesterone receptors, membrane receptors of the hormone such as membrane progesterone receptor (mPR) may also be involved in neuroprotection. Three mPR subtypes (mPRα, mPRß, and mPRγ) have been described and mPRα is best characterized pharmacologically. In the present study we investigated the distribution, cellular localization and the regulation of mPRα in male mouse and rat brain. We showed by reverse transcription-PCR that mPRα is expressed at similar levels in the male and female mouse brain suggesting that its expression may not be influenced by steroid levels. Treatment of males by estradiol or progesterone did not modify the level of expression of mPRα as shown by Western blot analysis. In situ hybridization and immunohistochemistry analysis showed a wide expression of mPRα in particular in the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, hippocampus and cerebellum. Double immunofluorescence and confocal microscopy analysis showed that mPRα is expressed by neurons but not by oligodendrocytes and astrocytes. In the rat brain, the distribution of mPRα was similar to that observed in mouse brain; and after TBI, mPRα expression was induced in oligodendrocytes, astrocytes and reactive microglia. The wide neuroanatomical distribution of mPRα suggests that this receptor may play a role beyond neuroendocrine and reproductive functions. However, in the absence of injury its role might be restricted to neurons. The induction of mPRα after TBI in microglia, astrocytes and oligodendrocytes, points to a potential role in mediating the modulatory effects of progesterone in inflammation, ion and water homeostasis and myelin repair in the injured brain.

Progesterone is neuroprotective against ischemic brain injury through its effects on the phosphoinositide 3-kinase/protein kinase B signaling pathway.

We tested the hypothesis that the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway mediates some of the neuroprotective effects of progesterone (PROG) after ischemic stroke. We examined whether PROG acting through the PI3K/Akt pathway could affect the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Rats underwent permanent focal cerebral ischemia by electrocoagulation and received intraperitoneal injections of PROG (8 mg/kg) or vehicle at 1 h post-occlusion and subcutaneous injections at 6, 24, and 48 h. PAkt/Akt levels, apoptosis and apoptosis-related proteins (phosphorylated Bcl-2-associated death promoter (pBAD), BAD, caspase-3, and cleaved caspase-3) were analyzed by TUNEL assays, Western blotting and immunohistochemistry at 24 h post-pMCAO. VEGF and BDNF were analyzed at 24, 72 h and 14 days post-pMCAO with Western blots. Following pMCAO, PROG treatment significantly (P<0.05) reduced ischemic lesion size and edema. Treatment with PROG significantly (P<0.05) decreased VEGF at 24 and 72 h but increased VEGF expression 14 days after injury. The treatment also increased BDNF, and attenuated apoptosis by increasing Akt phosphorylation compared with vehicle alone. The selective PI3K inhibitor wortmannin compromised PROG-induced neuroprotective effects and reduced the elevation of pAkt levels in the ischemic penumbra. Our findings lead us to suggest that the PI3K/Akt pathway can play a role in mediating the neuroprotective effects of PROG after stroke by altering the expression of trophic factors in the brain.

Progesterone in the treatment of acute traumatic brain injury: a clinical perspective and update.

Despite decades of laboratory research and clinical trials, a safe and effective treatment for traumatic brain injury has yet to reach clinical practice. The failure is due in part to the prevalence of a reductionist philosophy and research praxis that targets a single receptor mechanism, gene, or brain locus. This approach fails to account for the fact that traumatic brain injury is a very complex disease caused by a cascade of systemic toxic events in the brain and throughout the body. Attention is now turning to pleiotropic drugs that act on multiple genomic, proteomic, and metabolic pathways to enhance morphological and functional outcomes after brain injury. Of the agents now in clinical trial, the neurosteroid progesterone appears to hold considerable promise. Many still assume that progesterone is "just a female hormone" with limited, if any, neuroprotective properties, but this view is outdated. This review will survey the evidence that progesterone has salient pleiotropic properties as a neuroprotective agent in a variety of central nervous system injury models. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

The membrane-associated progesterone-binding protein 25-Dx: expression, cellular localization and up-regulation after brain and spinal cord injuries.

Progesterone has neuroprotective effects in the injured and diseased spinal cord and after traumatic brain injury (TBI). In addition to intracellular progesterone receptors (PR), membrane-binding sites of progesterone may be involved in neuroprotection. A first putative membrane receptor of progesterone, distinct from the classical intracellular PR isoforms, with a single membrane-spanning domain, has been cloned from porcine liver. Homologous proteins were cloned in rats (25-Dx), mice (PGRMC1) and humans (Hpr.6). We will refer to this progesterone-binding protein as 25-Dx. The distribution and regulation of 25-Dx in the nervous system may provide some clues to its functions. In spinal cord, 25-Dx is localized in cell membranes of dorsal horn neurons and ependymal cells lining the central canal. A role of 25-Dx in mediating the protective effects of progesterone in the spinal cord is supported by the observation that its mRNA and protein are up-regulated by progesterone in dorsal horn of the injured spinal cord. In contrast, the classical intracellular PRs were down-regulated under these conditions. In brain, 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, ependymal cells of the ventricular walls, and the meninges. Interestingly, it is co-expressed with vasopressin in neurons of the paraventricular, supraoptic and retrochiasmatic nuclei. In response to TBI, 25-Dx expression is up-regulated in neurons and induced in astrocytes. The expression of 25-Dx in structures involved in cerebrospinal fluid production and osmoregulation, and its up-regulation after brain damage, point to a potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after TBI. Our observations suggest that progesterone's actions may involve different signaling mechanisms depending on the pathophysiological context, and that 25-Dx may be involved in the neuroprotective effect of progesterone in the injured brain and spinal cord.

Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: analysis by gas chromatography/mass spectrometry.

Steroids in brain arise from the peripheral endocrine glands and local synthesis. In traumatic brain injury (TBI), the endogenous circulating hormones at the time of injury are important for neuroprotection. In particular, pseudopregnant females recover better than males from TBI. We investigated the effect of pseudopregnancy and TBI on steroid levels in plasma and in three brain regions (within, adjacent, and distal to the lesion site), 6 and 24 h after prefrontal cortex injury. The following steroids were analyzed by gas chromatography/mass spectrometry: pregnenolone, progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, 3beta,5alpha-tetrahydroprogesterone, dehydroepiandrosterone, Delta(4)-androstenedione, testosterone, 5alpha-dihydrotestosterone, 3alpha,5alpha-tetrahydrotestosterone, 3beta,5alpha-tetrahydrotestosterone, and 17beta-estradiol. Corticosterone was assayed in plasma to account for stress in the rats. We found different steroid profiles in brain and plasma of male and pseudopregnant female rats and specific profile changes after TBI. In sham-operated pseudopregnant females, much higher levels of progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, and 3beta,5alpha-tetrahydroprogesterone were measured in both brain and plasma, compared with sham-operated males. Plasma levels of corticosterone were high in all groups, indicating that the surgeries induced acute stress. Six hours after TBI, the levels of pregnenolone, progesterone, and 5alpha-dihydroprogesterone increased, and those of testosterone decreased in male brain, whereas levels of 5alpha-dihydroprogesterone and 3beta,5alpha-tetrahydroprogesterone increased in brain of pseudopregnant female rats. Plasma levels of 5alpha-dihydroprogesterone did not change after TBI, suggesting a local activation of the 5alpha-reduction pathway of progesterone in both male and pseudopregnant female brain. The significant increase in neurosteroid levels in the male brain after TBI is consistent with their role in neuroprotection. In pseudopregnant females, high levels of circulating progestagens may provide protection against TBI.

Allopregnanolone and progesterone decrease cell death and cognitive deficits after a contusion of the rat pre-frontal cortex.

We compared the effects of three different doses of allopregnanolone (4, 8 or 16 mg/kg), a metabolite of progesterone, to progesterone (16 mg/kg) in adult rats with controlled cortical impact to the pre-frontal cortex. Injections were given 1 h, 6 h and every day for 5 consecutive days after the injury. One day after injury, both progesterone-treated (16 mg/kg) and allopregnanolone (8 or 16 mg/kg)-treated rats showed less caspase-3 activity, and rats treated with allopregnanolone (16 mg/kg) showed less DNA fragmentation in the lesion area, indicating reduced apoptosis. Nineteen days after the injury, rats treated with progesterone and allopregnanolone (8 or 16 mg/kg) showed no difference in necrotic cavity size but had less cell loss in the medio-dorsal nucleus of the thalamus and less learning and memory impairments compared with the injured vehicle-treated rats. On that same day the injured rats treated with progesterone showed more weight gain compared with the injured rats treated with the vehicle. These results can be taken to show that progesterone and allopregnanolone have similar neuroprotective effects after traumatic brain injury, but allopregnanolone appears to be more potent than progesterone in facilitating CNS repair.

Serum progesterone levels correlate with decreased cerebral edema after traumatic brain injury in male rats.

Previous animal research suggests that progesterone may have powerful neuroprotective effects in traumatic brain injury (TBI). This experiment tested the hypothesis that progesterone levels correlate with decreased cerebral edema in male rats with bilateral medial frontal cortex injuries. Three groups of male Sprague-Dawley rats were used: injured given progesterone (4 mg/kg), injured given vehicle (oil), and uninjured controls given vehicle. Progesterone or vehicle was administered intraperitoneally at 1, 6, and 24 h postinjury. At 48 h postinjury, the rats were killed, brains extracted, and assayed for edema. Percent difference in water content of the area surrounding the lesion was compared to posterior cortex. A strong inverse relationship was found between serum progesterone levels and percent cerebral edema; the higher the progesterone levels, the lower the percent edema. Both progesterone and oil-treated animals had some edema compared to sham-operated controls. The brains of the injured animals given control solution were higher in water content than either the uninjured group or injured progesterone-treated rats 48 h postinjury. These findings confirm that progesterone significantly decreases cerebral edema after TBI in adult male subjects.

Brain damage, sex hormones and recovery: a new role for progesterone and estrogen?

Estrogen and progesterone, long considered for their roles as primary hormones in reproductive and maternal behavior, are now being studied as neuroprotective and neuroregenerative agents in stroke and traumatic brain injuries. Collectively, the hormones reduce the consequences of the injury cascade by enhancing anti-oxidant mechanisms, reducing excitotoxicity (altering glutamate receptor activity, reducing immune inflammation, providing neurotrophic support, stimulating axonal remyelinization), and enhancing synaptogenesis and dendritic arborization. Estrogen seems more effective as a prophylactic treatment in females at risk for cardiac and ischemic brain injury, whereas progesterone appears to be more helpful in the post-injury treatment of both male and female subjects with acute traumatic brain damage. However, a recent clinical trial with estradiol replacement therapy in elderly women that have a history of cerebrovascular disease, showed that this hormone was unable to protect against reoccurrence of ischemia or to reduce the incidence of mortality compared to a placebo.

Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury.

Tissue engineering in the post-injury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. We have characterized the use of methylcellulose (MC) as a scaffolding material, whose concentration and solvent were varied to manipulate its physical properties. MC solutions were produced to exhibit low viscosity at 23 degrees C and form a soft gel at 37 degrees C, thereby making MC attractive for minimally invasive procedures in vivo. Degradation and swelling studies in vitro demonstrated a small amount of initial polymer erosion followed by relative polymer stability over the 2-week period tested as well as increased hydrogel mass due to solvent uptake. Concentrations up to 8% did not elicit cell death in primary rat astrocytes or neurons at 1 or 7 days. Acellular 2% MC (30 microl) was microinjected into the brains of rats 1 week after cortical impact injury (velocity = 3 m/s, depth = 2 mm) and examined at 2 days (n = 8; n = 3, vehicle injected) and 2 weeks (n = 5; n = 3, vehicle injected). The presence of MC did not alter the size of the injury cavity or change the patterns of gliosis as compared to injured, vehicle-injected rats (detected using antibodies against GFAP and ED1). Collectively, these data indicate that MC is well suited as a biocompatible injectable scaffold for the repair of defects in the brain.

Behavioral and morphological consequences of primary astrocytes transplanted into the rat cortex immediately after nucleus basalis ibotenic lesion.

Adult male rats received transplants of dissociated 30-day old cultured cortical astrocytes into the ipsilateral frontal and parietal cortex immediately after unilateral ibotenic acid lesion of the NBM or after sham injury. We hypothesized that transplants of astrocytes into the acetylcholine-deprived cortex might provide trophic support to terminals arising from damaged NBM neurons. Twenty four hours after transplantation and every other day for 11 days post surgery, the animals were tested for locomotion and habituation in an open field. NBM lesion reduced vertical movements only as compared to no lesion and no transplant counterparts. Nine days after surgery rats with NBM lesion and astrocyte-transplants into the cortex were as impaired in the acquisition of a passive avoidance (PA) task as untreated counterparts. Animals with no lesions and transplants into the cortex also had significant PA acquisition deficits. All rats with ibotenic lesion were significantly impaired on PA retention as compared to rats with no lesions. Astrocyte-transplants survived up to 2 months after cortical implantation but these transplants produced severe laminar disruption and gliosis. This effect was greater in rats with NBM lesion than in intact animals with transplants into the cortex. These data show that astrocyte-transplants do not promote functional recovery after NBM lesion and suggest an immune rejection of the astrocyte transplants by the host brain.

Effects of the duration of progesterone treatment on the resolution of cerebral edema induced by cortical contusions in rats.

UNLABELLED: PURPOSE. The aim of the present study was to assess the effect of different durations of administration of progesterone (4 mg/kg) on the resolution of edema 6 days after medial frontal cortex contusions (MFC) in male adult rats. METHODS: Animals sustaining injury were injected with progesterone or its vehicle for 3 days or for 5 days beginning the first hour after surgery. On the 6th day the rats were killed and their brain water content was measured. RESULTS: We confirmed the presence of edema six days after MFC. However, both 3 and 5 days of treatment with progesterone significantly reduced edema in the injured brains but the five days of treatment were more effective. The effects of progesterone depend upon the duration of the treatment because there are two waves of edema. The first phase begins within a few hours of the injury and the second starts several days later. CONCLUSIONS: Our data are consistent with earlier findings showing that longer durations of progesterone administration lead to more complete behavioral recovery as well as to an increased number of surviving neurons.

Does endogenous progesterone promote recovery of chronic sensorimotor deficits following contusion to the forelimb representation of the sensorimotor cortex?

We studied sensorimotor recovery in male, normal-cycling and pseudopregnant female rats following unilateral FL-SMC contusions. Forelimb use (push off before a rear, support against the walls, and landing after a rear) and the foot fault test (foot misplacements during locomotion on an elevated grid) were analyzed from videotapes taken before surgery, and then again on post-surgical days 2 and 36. High endogenous progesterone levels in females at the time of injury did not affect recovery as there were no differences between males, pseudopregnant females and normal-cycling female rats on these behaviors. None of the brain-injured rats recovered symmetrical forelimb use between 2 and 36 days after injury (P>0.05) and they also showed foot misplacements (P>0.05) in the foot fault test. Male and female rats with contusions had fewer mean foot misplacements on day 36 than 2 days after injury (P<0.001), indicating that there was partial recovery on this task. These results were taken to show that there were no sex differences in motor deficits caused by unilateral FL-SMC injury. In addition, higher endogenous progesterone levels in females did not protect them from the chronic sensorimotor deficits caused by unilateral FL-SMC contusions.

Effects of the novel NMDA receptor antagonist gacyclidine on recovery from medial frontal cortex contusion injury in rats.

Gacyclidine, a novel, noncompetitive NMDA receptor antagonist, was injected (i.v.) into rats at three different doses to determine if the drug could promote behavioral recovery and reduce the behavioral and anatomical impairments that occur after bilateral contusions of the medial frontal cortex (MFC). In the Morris water maze, contused rats treated with gacyclidine at a dosage of 0.1 mg/kg performed better than their vehicle-treated conspecifics. Rats given gacyclidine at either 0.3 or 0.03 mg/kg performed better than brain-injured controls, but not as well as those treated with 0.1 mg/kg. Counts of surviving neurons in the nucleus basalis magnocellularis (NBM) and the medial dorsal nucleus (MDN) of the thalamus were used to determine whether gacyclidine treatment attenuated secondary cell death. In both the NBM and the MDN, the counts revealed fewer surviving neurons in untreated contused rats than in gacyclidine-treated rats. Increases in the size and number of microglia and astrocytes were observed in the striatum of gacyclidine-treated contused brains. Although most consequences of MFC contusions were attenuated, we still observed increases in ventricle dilation and thinning of the cortex. In fact, the ventricles of rats treated with 0.1 mg/kg of gacyclidine were larger than those of their vehicle treated counterparts, although we observed no behavioral impairment.

The effect of Ginkgo biloba extract (EGb 761) on gliotic reactions in the hippocampal formation after unilateral entorhinal cortex lesions.

PURPOSE: Ginkgo biloba extract (EGb 761) has been shown to facilitate behavioral and neuro-morphological recovery from brain injury, but less is known about its effects on glia. Since gliosis may be an important component of the recovery process, we tested the hypothesis that EGb 761 alters the time course and development of microglial activation and astrocytosis after brain injury. METHODS: Rats were treated with either saline or EGb 761 and killed at 2 hrs, 1, 3, 7, and 14 days following unilateral entorhinal cortex (EC) lesions. Microglia and their precursors were visualized with a silver impregnation method, and astrocytes with GFAP. RESULTS: Blood-borne monocytes/macrophages were seen as early as 2 hrs after injury in all animals. The side contralateral to the injury showed minimal microglial activation and there were no significant effects of drug treatment. On the side ipsilateral to the lesion EGb 761 enhanced microglial activation at 3, 7, and 14 days in the molecular layer and the hilus of the dentate gyrus; the areas of most profound deaf-ferentation after EC injury. Regions of the corpus callosum also showed enhanced microglial activation over the same time course. Reactive astrocytes were stained with GFAP and were found to be more numerous than activated microglia, particularly in the ipsilateral corpus callo-sum. EGb 761 treatment enhanced astrocytosis at 3 days in the molecular layer, the hilus, and the corpus callosum on the ipsilateral side. CONCLUSIONS: Taken together our results show that EGb 761 enhances, accelerates and prolongs the activation of microglia and astrocytosis at the site of injury.

GM1 produces attenuation of short-term memory deficits in Hebb-Williams maze performance after unilateral entorhinal cortex lesions.

The Hebb-Williams maze was used to examine spatial abilities of adult male Sprague-Dawley rats with unilateral electrolytic entorhinal cortex lesions. The injured rats were treated for 14 days with either saline or ganglioside GM1. Testing was begun 7 weeks following injury, and involved 12 maze problems with independent configurations, with immediate starting replacement used for the six trials per problem. Compared to sham-operated counterparts, the rats with lesion plus saline treatment were impaired in total number of errors, initial entry errors, and repeat errors over 12 consecutive problems. GM1-treated rats showed improved performance, making significantly fewer total and repeat errors, indicating that this substance may be potentially useful as therapy after entorhinal cortex injury.

Regional changes in the expression of neurotrophic factors and their receptors following acute traumatic brain injury in the adult rat brain.

We have analyzed the effect of severe traumatic brain injury (TBI) on the levels of mRNA expression of neurotrophic factors (NTFs): brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and their respective receptors: trkB, trkA and CNTFRalpha. The expression was examined in the region of the lesion as well as a region remote from the lesion at 12, 24, and 36 h following the injury. Our data suggest that after the brain injury, the expression of NGF and BDNF mRNAs were early, transiently and significantly upregulated while that of CNTF was a slow and less amplified response in both areas of the brain. We also found that trkA mRNA expression was only upregulated significantly in the remote area; trkB mRNA showed no significant change in either area except an upregulation at 12 h in the remote area. CNTFRalpha was downregulated significantly by 24-36 h in the lesion area and by 24 h in the remote area. These changes suggest that TBI regulates the expression of NTFs and their receptors. These alterations in expression may be involved in modulating the neuronal response after brain injury.

Effects of unilateral entorhinal cortex lesion on retention of water maze performance.

In a previous study, adult male Sprague-Dawley rats with unilateral, electrolytic entorhinal cortex lesions showed significant deficits in acquisition of a water maze task that measured working memory. The 10 days of testing used two trials per day with an intertrial interval of 1 h, and the rats with entorhinal damage were impaired in total distance to the platform in both trials. In the present retention study, rats who learned the same task prior to injury and were then retested for 5 days after lesion showed only a first day deficit in total distance to platform in the second trial. Analysis of swim patterns indicated that rats with unilateral entorhinal lesions used an altered strategy in retention testing to find the platform in the second trial of each day and incorporated the use of headings appropriate for Trial 1 only. This altered or compensatory strategy was not the optimum choice for problem solution. Although the rats then were able to switch headings and find the platform without significant impairment in total distance to platform on days 2-5 of testing, the use of an initial incorrect strategy indicated subtle residual deficits in cue integration and use of working memory.