The paradox of tPA in ischemic stroke: tPA knockdown following recanalization improves functional and histological outcomes.

Previous studies have demonstrated that endogenous tissue-type plasminogen activator (tPA) is upregulated in the brain after an acute ischemic stroke (AIS). While mixed results were observed in genetic models, the pharmacological inhibition of endogenous tPA showed beneficial effects. Treatment with exogenous recombinant tPA exacerbated brain damage in rodent models of stroke. Despite the detrimental effects of tPA in ischemic stroke, recombinant tPA is administered to AIS patients to recanalize the occluded blood vessels because the benefits of its administration outweigh the risks associated with tPA upregulation and increased activity. We hypothesized that tPA knockdown following recanalization would ameliorate sensorimotor deficits and reduce brain injury. Young male and female rats (2-3 months old) were subjected to transient focal cerebral ischemia by occlusion of the right middle cerebral artery. Shortly after reperfusion, rats from appropriate cohorts were administered a nanoparticle formulation containing tPA shRNA or control shRNA plasmids (1 mg/kg) intravenously via the tail vein. Infarct volume during acute and chronic phases, expression of matrix metalloproteinases (MMPs) 1, 3, and 9, enlargement of cerebral ventricle volume, and white matter damage were all reduced by shRNA-mediated gene silencing of tPA following reperfusion. Additionally, recovery of somatosensory and motor functions was improved. In conclusion, our results provide evidence that reducing endogenous tPA following recanalization improves functional outcomes and reduces post-stroke brain damage.

The Impact of Social Isolation and Environmental Deprivation on Blood Pressure and Depression-Like Behavior in Young Male and Female Mice.

Background: Social isolation (SI) and loneliness are major adult and adolescent health concerns, particularly in the coronavirus disease 2019 (COVID-19) era. Recent prospective cohort studies indicate that older women who experienced both SI and loneliness had a significantly higher risk of cardiovascular disease (CVD). Hypertension, a well-established risk factor for CVD, is more prevalent in elderly women than men. Furthermore, a lack of social relationships is strongly associated with an increased risk of hypertension in middle-aged and elderly women compared to men. Although this has not been extensively studied, adolescents and young adults who experience loneliness or SI may also be at risk for CVD and depression. The purpose of this study was to examine the effect of SI on blood pressure and depression-like behavior in young male and female mice. Methods: Weaned C57BL/6 mice were randomly assigned (n = 6/group/sex) to either group housing (GH) or SI. Animals in the SI group were housed in individual cages for 8 weeks with no view of other animals. The cages were kept in ventilated racks to prevent pheromone exposure and socially isolated animals had no cage enrichment. Results: SI increased systolic, diastolic, and mean arterial blood pressure in females and elevated heart rate in both sexes. Body weight gain was dramatically increased in socially isolated females but tended to decrease in socially isolated males. In the forced swim test, which detects depression-like behavior, there was no difference between groups in total immobility time. The latency to immobility, however, was significantly decreased in socially isolated females. Serum concentrations of corticosterone and metanephrine did not differ between socially isolated and group-housed females, but corticosterone levels were significantly reduced in socially isolated males. Conclusions: Our results indicate that 8 weeks of SI leads to significant changes in blood pressure and heart rate and mild changes in depression-like behavior in young mice, with females affected more than males.

The interplay between MMP-12 and t-PA in the brain after ischemic stroke.

Tissue-type plasminogen activator (t-PA) expression is known to increase following transient focal cerebral ischemia and reperfusion. Previously, we reported downregulation of t-PA upon suppression of matrix metalloproteinase-12 (MMP-12), following transient focal cerebral ischemia and reperfusion. We now present data on the temporal expression of t-PA in the brain after transient ischemia, as well as the interaction between MMP-12 and t-PA, two proteases associated with the breakdown of the blood-brain barrier (BBB) and ischemic brain damage. We hypothesized that there might be reciprocal interactions between MMP-12 and t-PA in the brain after ischemic stroke. This hypothesis was tested using shRNA-mediated gene silencing and computational modeling. Suppression of t-PA following transient ischemia and reperfusion in rats attenuated MMP-12 expression in the brain. The overall effect of t-PA shRNA administration was to attenuate the degradation of BBB tight junction protein claudin-5, diminish BBB disruption, and reduce neuroinflammation by decreasing the expression of the microglia/macrophage pro-inflammatory M1 phenotype (CD68, iNOS, IL-1ß, and TNFα). Reduced BBB disruption and subsequent lack of infiltration of macrophages (the main source of MMP-12 in the ischemic brain) could account for the decrease in MMP-12 expression after t-PA suppression. Computational modeling of in silico protein-protein interactions indicated that MMP-12 and t-PA may interact physically. Overall, our findings demonstrate that MMP-12 and t-PA interact directly or indirectly at multiple levels in the brain following an ischemic stroke. The present findings could be useful in the development of new pharmacotherapies for the treatment of stroke.

Therapeutic efficacy of matrix metalloproteinase-12 suppression on neurological recovery after ischemic stroke: Optimal treatment timing and duration.

We recently showed that the post-ischemic induction of matrix metalloproteinase-12 (MMP-12) in the brain degrades tight junction proteins, increases MMP-9 and TNFα expression, and contributes to the blood-brain barrier (BBB) disruption, apoptosis, demyelination, and infarct volume development. The objectives of this study were to (1) determine the effect of MMP-12 suppression by shRNA-mediated gene silencing on neurological/functional recovery, (2) establish the optimal timing of MMP-12shRNA treatment that provides maximum therapeutic benefit, (3) compare the effectiveness of acute versus chronic MMP-12 suppression, and (4) evaluate potential sex-related differences in treatment outcomes. Young male and female Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats were administered either MMP-12shRNA or scrambled shRNA sequence (control) expressing plasmids (1 mg/kg; i.v.) formulated as nanoparticles. At designated time points after reperfusion, rats from various groups were subjected to a battery of neurological tests to assess their reflex, balance, sensory, and motor functions. Suppression of MMP-12 promoted the neurological recovery of stroke-induced male and female rats, although the effect was less apparent in females. Immediate treatment after reperfusion resulted in a better recovery of sensory and motor function than delayed treatments. Chronic MMP-12 suppression neither enhanced nor diminished the therapeutic effects of acute MMP-12 suppression, indicating that a single dose of plasmid may be sufficient. We conclude that suppressing MMP-12 after an ischemic stroke is a promising therapeutic strategy for promoting the recovery of neurological function.

Stem cell treatment improves post stroke neurological outcomes: a comparative study in male and female rats.

BACKGROUND AND PURPOSE: The therapeutic potential of different stem cells for ischaemic stroke treatment is intriguing and somewhat controversial. Recent results from our laboratory have demonstrated the potential benefits of human umbilical cord blood-derived mesenchymal stem cells (MSC) in a rodent stroke model. We hypothesised that MSC treatment would effectively promote the recovery of sensory and motor function in both males and females, despite any apparent sex differences in post stroke brain injury. METHODS: Transient focal cerebral ischaemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery. Following the procedure, male and female rats of the untreated group were euthanised 1 day after reperfusion and their brains were used to estimate the resulting infarct volume and tissue swelling. Additional groups of stroke-induced male and female rats were treated with MSC or vehicle and were subsequently subjected to a battery of standard neurological/neurobehavioral tests (Modified Neurological Severity Score assessment, adhesive tape removal, beam walk and rotarod). The tests were administered at regular intervals (at days 1, 3, 5, 7 and 14) after reperfusion to determine the time course of neurological and functional recovery after stroke. RESULTS: The infarct volume and extent of swelling of the ischaemic brain were similar in males and females. Despite similar pathological stroke lesions, the clinical manifestations of stroke were more pronounced in males than females, as indicated by the neurological scores and other tests. MSC treatment significantly improved the recovery of sensory and motor function in both sexes, and it demonstrated efficacy in both moderate stroke (females) and severe stroke (males). CONCLUSIONS: Despite sex differences in the severity of post stroke outcomes, MSC treatment promoted the recovery of sensory and motor function in male and female rats, suggesting that it may be a promising treatment for stroke.

Attenuation of the Induction of TLRs 2 and 4 Mitigates Inflammation and Promotes Neurological Recovery After Focal Cerebral Ischemia.

The intense inflammatory response triggered in the brain after focal cerebral ischemia is detrimental. Recently, we showed that the suppression of toll-like receptors (TLRs) 2 and 4 attenuates infarct size and reduces the expression of pro-inflammatory cytokines in the ischemic brain. In this study, we further examined the effect of unsuppressed induction of TLRs 2 and 4 on the expression of its downstream signaling molecules and pro-inflammatory cytokines 1 week after reperfusion. The primary purpose of this study was to investigate the effect of simultaneous knockdown of TLRs 2 and 4 on M1/M2 microglial polarization dynamics and post-stroke neurological deficits and the recovery. Transient focal cerebral ischemia was induced in young adult male Sprague-Dawley rats by the middle cerebral artery occlusion (MCAO) procedure using a monofilament suture. Appropriate cohorts of rats were treated with a nanoparticle formulation of TLR2shRNA and TLR4shRNA (T2sh+T4sh) expressing plasmids (1 mg/kg each of T2sh and T4sh) or scrambled sequence inserted vector (vehicle control) expressing plasmids (2 mg/kg) intravenously via tail vein immediately after reperfusion. Animals from various cohorts were euthanized during reperfusion, and the ischemic brain tissue was isolated and utilized for PCR followed by agarose gel electrophoresis, real-time PCR, immunoblot, and immunofluorescence analysis. Appropriate groups were subjected to a battery of standard neurological tests at regular intervals until 14 days after reperfusion. The increased expression of both TLRs 2 and 4 and their downstream signaling molecules including the pro-inflammatory cytokines was observed even at 1-week after reperfusion. T2sh+T4sh treatment immediately after reperfusion attenuated the post-ischemic inflammation, preserved the motor function, and promoted recovery of the sensory and motor functions. We conclude that the post-ischemic induction of TLRs 2 and 4 persists for at least 7 days after reperfusion, contributes to the severity of acute inflammation, and impedes neurological recovery. Unlike previous studies in TLRs 2 or 4 knockout models, results of this study in a pharmacologically relevant preclinical rodent stroke model have translational significance.

Early postnatal manganese exposure causes arousal dysregulation and lasting hypofunctioning of the prefrontal cortex catecholaminergic systems.

Studies have reported associations between environmental manganese (Mn) exposure and impaired cognition, attention, impulse control, and fine motor function in children. Our recent rodent studies established that elevated Mn exposure causes these impairments. Here, rats were exposed orally to 0, 25, or 50 mg Mn kg-1  day-1 during early postnatal life (PND 1-21) or lifelong to determine whether early life Mn exposure causes heightened behavioral reactivity in the open field, lasting changes in the catecholaminergic systems in the medial prefrontal cortex (mPFC), altered dendritic spine density, and whether lifelong exposure exacerbates these effects. We also assessed astrocyte reactivity (glial fibrillary acidic protein, GFAP), and astrocyte complement C3 and S100A10 protein levels as markers of A1 proinflammatory or A2 anti-inflammatory reactive astrocytes. Postnatal Mn exposure caused heightened behavioral reactivity during the first 5-10 min intervals of daily open field test sessions, consistent with impairments in arousal regulation. Mn exposure reduced the evoked release of norepinephrine (NE) and caused decreased protein levels of tyrosine hydroxylase (TH), dopamine (DA) and NE transporters, and DA D1 receptors, along with increased DA D2 receptors. Mn also caused a lasting increase in reactive astrocytes (GFAP) exhibiting increased A1 and A2 phenotypes, with a greater induction of the A1 proinflammatory phenotype. These results demonstrate that early life Mn exposure causes broad lasting hypofunctioning of the mPFC catecholaminergic systems, consistent with the impaired arousal regulation, attention, impulse control, and fine motor function reported in these animals, suggesting that mPFC catecholaminergic dysfunction may underlie similar impairments reported in Mn-exposed children.

Early Postnatal Manganese Exposure Reduces Rat Cortical and Striatal Biogenic Amine Activity in Adulthood.

Growing evidence from studies with children and animal models suggests that elevated levels of manganese during early development lead to lasting cognitive and fine motor deficits. This study was performed to assess presynaptic biogenic amine function in forebrain of adult Long-Evans rats exposed orally to 0, 25, or 50 mg Mn/kg/day over postnatal day 1-21 or continuously from birth to the end of the study (approximately postnatal day 500). Intracerebral microdialysis in awake rats quantified evoked outflow of biogenic amines in the right medial prefrontal cortex and left striatum. Results indicated that brain manganese levels in the early life exposed groups (postnatal day 24) largely returned to control levels by postnatal day 66, whereas levels in the lifelong exposed groups remained elevated 10%-20% compared with controls at the same ages. Manganese exposure restricted to the early postnatal period caused lasting reductions in cortical potassium-stimulated extracellular norepinephrine, dopamine, and serotonin, and reductions in striatal extracellular dopamine. Lifelong manganese exposure produced similar effects with the addition of significant decreases in cortical dopamine that were not evident in the early postnatal exposed groups. These results indicate that early postnatal manganese exposure produces persistent deficits in cortical and striatal biogenic amine function. Given that these same animals exhibited lasting impairments in attention and fine motor function, these findings suggest that reductions in catecholaminergic activity are a primary factor underlying the behavioral effects caused by manganese, and indicate that children exposed to elevated levels of manganese during early development are at the greatest risk for neuronal deficiencies that persist into adulthood.

Oral methylphenidate alleviates the fine motor dysfunction caused by chronic postnatal manganese exposure in adult rats.

Developmental manganese (Mn) exposure is associated with motor dysfunction in children and animal models, but little is known about the underlying neurochemical mechanisms or the potential for amelioration by pharmacotherapy. We investigated whether methylphenidate (MPH) alleviates fine motor dysfunction due to chronic postnatal Mn exposure, and whether Mn exposure impairs brain extracellular dopamine (DA) and norepinephrine (NE) in the prefrontal cortex (PFC) and striatum in adult animals. Rats were orally exposed to 0 or 50 mg Mn/kg/day from postnatal day 1 until the end of the study (PND 145). The staircase test was used to assess skilled forelimb function. Oral MPH (2.5 mg/kg/day) was administered daily 1 h before staircase testing for 16 days. DA and NE levels were measured by dual probe microdialysis. Results show that Mn exposure impaired reaching and grasping skills and the evoked release of DA and NE in the PFC and striatum of adult rats. Importantly, oral MPH treatment fully alleviated the fine motor deficits in the Mn-exposed animals, but did not affect forelimb skills of control rats not exposed to Mn. These results suggest that catecholaminergic hypofunctioning in the PFC and striatum may underlie the Mn-induced fine motor dysfunction, and that oral MPH pharmacotherapy is an effective treatment approach for alleviating this dysfunction in adult animals. The therapeutic potential of MPH for the treatment of motor dysfunction in Mn-exposed children and adults appears promising pending further characterization of MPH efficacy in other functional areas (eg, attention) believed to be affected by developmental Mn exposure.

Activity-based anorexia is associated with reduced hippocampal cell proliferation in adolescent female rats.

Activity-based anorexia (ABA) is an animal model of anorexia nervosa that mimics core features of the clinical psychiatric disorder, including severe food restriction, weight loss, and hyperactivity. The ABA model is currently being used to study starvation-induced changes in the brain. Here, we examined hippocampal cell proliferation in animals with ABA (or the appropriate control conditions). Adolescent female Sprague-Dawley rats were assigned to 4 groups: control (24h/day food access), food-restricted (1h/day food access), exercise (24h/day food and wheel access), and ABA (1h/day food access, 24h/day wheel access). After 3 days of ABA, 5-bromo-2'-deoxyuridine (BrdU; 200mg/kg, i.p.) was injected and the rats were perfused 2h later. Brains were removed and subsequently processed for BrdU and Ki67 immunohistochemistry. The acute induction of ABA reduced cell proliferation in the dentate gyrus. This effect was significant in the hilus region of the dentate gyrus, but not in the subgranular zone, where adult neurogenesis occurs. Marked decreases in cell proliferation were also observed in the surrounding dorsal hippocampus and in the corpus callosum. These results indicate a primary effect on gliogenesis rather than neurogenesis following 3 days of ABA. For each brain region studied (except SGZ), there was a strong positive correlation between the level of cell proliferation and body weight/food intake. Future studies should examine whether these changes are maintained following long-term weight restoration and whether alterations in neurogenesis occur following longer exposures to ABA.

The appropriateness of unbiased optical fractionators to assess cell proliferation in the adult hippocampus.

Optical fractionators have dominated the field of neural cell counting for two decades. These unbiased stereological techniques are often used for the quantification of hippocampal cell proliferation in neurogenesis experiments. However, the heterogeneous distribution of labeled cells, especially in the form of clusters, confounds the application of these techniques. A critical evaluation of the applicability of the optical fractionator suggests that absolute counting achieves higher efficiency in the quantification of cell proliferation than unbiased estimations.

Tobacco smoke diminishes neurogenesis and promotes gliogenesis in the dentate gyrus of adolescent rats.

Brain disorders and environmental factors can affect neurogenesis and gliogenesis in the hippocampus. These studies investigated the effects of chronic exposure to tobacco smoke on progenitor cell proliferation and the survival and phenotype of new cells in the dentate gyrus of adolescent rats. The rats were exposed to tobacco smoke for 4h/day for 14 days. To investigate cell proliferation, the exogenous marker 5-bromo-2'-deoxyuridine (BrdU, 200mg/kg, ip) was administered 2h into the 4-h smoke exposure session on day 14. The rats were sacrificed 2-4h after the administration of BrdU. To investigate cell survival, the same dose of BrdU was administered 24h before the start of the 14-day smoke exposure period. These rats were sacrificed 24h after the last smoke exposure session. Tobacco smoke exposure decreased both the number of dividing progenitor cells (-19%) and the number of surviving new cells (-20%), labeled with BrdU in the dentate gyrus. The decrease in cell proliferation was not associated with an increase in apoptotic cell death, as shown by TUNEL analysis. Colocalization studies indicated that exposure to tobacco smoke decreased the number of new immature neurons (BrdU/DCX-positive) and transition neurons (BrdU/DCX/NeuN-positive) and increased the number of new glial cells (BrdU/GFAP-positive). These findings demonstrate that exposure to tobacco smoke diminishes neurogenesis and promotes gliogenesis in the dentate gyrus of adolescent rats. These effects may play a role in the increased risk for depression and cognitive impairment in adolescent smokers.

Suppression of hippocampal cell proliferation by short-term stimulant drug administration in adult rats.

Sleep loss is known to potently suppress adult hippocampal cell proliferation and neurogenesis. Whether sleep suppression following acute administration of stimulant drugs also decreases hippocampal cell proliferation is not known. The present study examined the effect of three mechanistically distinct stimulants (caffeine, methamphetamine and modafinil) on cell proliferation. To maximize sleep suppression, these drugs were administered to rats (three i.p. injections, once every 4 h) during their sleep period (i.e. 12-h light phase). At the end of the light phase, 5-bromo-2'-deoxyuridine (200 mg/kg, i.p.) was injected and animals were killed 2 h later. Polygraphic recordings and locomotor activity measurements confirmed the wake-promoting and sleep-suppressing actions of each treatment. Results indicate that caffeine (20 mg/kg), methamphetamine (1.5 mg/kg) and modafinil (300 mg/kg) differentially suppressed sleep (45-91%) and selectively reduced cell proliferation in the hilus (12-44%), these results being significant for both caffeine and modafinil. When the same experiment was repeated in the dark (active) phase, the suppressant effect on hippocampal cell proliferation was either absent or greatly attenuated. In a further experiment, the effect of acute modafinil treatment in the light phase was shown to persist for 3 weeks after BrdU administration. We hypothesize that the differential effect of the stimulant drugs in the light vs. dark phase is attributable primarily to sleep suppression in the light. As abuse of stimulant drugs invariably leads to disrupted sleep in humans, our results suggest that they may, at least in part, decrease hippocampal neurogenesis via sleep loss and thereby adversely affect hippocampal-dependent processes.

Effects of the hypnotic drug zolpidem on cell proliferation and survival in the dentate gyrus of young and old rats.

Sleep loss/disruption has been shown to suppress adult hippocampal neurogenesis. Whether the administration of hypnotic drugs, by promoting sleep, especially in older subjects, who typically exhibit poor sleep, has a beneficial effect on neurogenesis parameters is unknown. We examined the effects of zolpidem, a widely prescribed nonbenzodiazepine hypnotic, on cell proliferation and survival in the dentate gyrus of young ( approximately 2 1/2 months) and old ( approximately 13 months) male Sprague-Dawley rats. Zolpidem (5, 10 or 20 mg/kg, i.p.) or vehicle was administered twice daily, at the beginning and middle of the sleep period, for either 2 days (acute study) or 21 days (chronic study). Proliferation and cell survival were measured by staining for Ki67 or 5-bromo-2'-deoxyurdine (BrdU), respectively. Acute administration of zolpidem produced a suppression of cell proliferation, which attained statistical significance only in the aged animals. The magnitude of the suppressive effect was larger in the hilus than in the subgranular zone (SGZ). In contrast, chronic administration of zolpidem produced little or no effect on proliferation in either age group, despite marked differences in basal proliferation levels between the two age groups. Similarly, there was little change in cell survival following chronic zolpidem administration in young versus old animals. A slight reduction of cell survival in the granular cell layer (GCL)/SGZ was observed in young animals and a slight augmentation in aged animals. To the extent that zolpidem improves sleep, these data suggest little or no benefit of hypnotic drug treatment on neurogenesis parameters in young or old rats.

Age-dependent decline in hippocampal neurogenesis is not altered by chronic treatment with fluoxetine.

There has been ongoing controversy as to whether selective serotonin reuptake inhibitors (SSRIs) exhibit the same antidepressant efficacy and risk profile within different age groups. Although the etiology of such potential differences is currently not clear, age-dependent differences in the rate of hippocampal neurogenesis offer one possibility. In the current studies we have therefore examined whether fluoxetine, the prototypical selective serotonin reuptake inhibitor, differentially modulates neurogenesis in adolescent, young adult, and aged rats. Proliferation in the dentate gyrus was measured by assaying expression of the endogenous proliferative marker, Ki67. Survival of proliferating cells was assayed by staining with BrdU. We confirmed previous reports that the rate of neurogenesis, as well as the survival of proliferating cells, decreases significantly with age. Moderate decreases were found in young adult rats relative to adolescent rats, and profound decreases were found in aged rats. We additionally found that age did not alter the response to 25 days of treatment with fluoxetine. In fact, we did not observe enhancement of hippocampal neurogenesis, nor enhancement of proliferating cell survival, in any of the three age groups despite using doses of fluoxetine which have been reported to be effective. In addition to finding no age-dependent effects, our data question the general reproducibility of previously reported fluoxetine effects in animals.

The effect of prolonged anesthesia with isoflurane, propofol, dexmedetomidine, or ketamine on neural cell proliferation in the adult rat.

BACKGROUND: Recent evidence indicates that new neurons are produced in the adult hippocampus, and play a functional role in cognitive processes such as learning and memory. In animals, new neuron production is suppressed by increasing age, gamma-aminobutyric acid receptor activity, reductions in basal forebrain activity and brain norepinephrine levels, and decreased environmental stimuli. Similarities between these effects and those of anesthetic administration suggest that anesthetics may modulate new cell production, and raise the possibility that postoperative cognitive dysfunction may result, in part, from anesthetic-induced suppression of adult neurogenesis. To test this hypothesis, we investigated the effects of prolonged anesthesia with four different anesthetics on hippocampal cell proliferation in young and older rats. METHODS: Young (approximately 3 mo) and older, middle-aged (approximately 12 mo) male Sprague-Dawley rats received one of four anesthetics (propofol, isoflurane, dexmedetomidine, and ketamine) for 8 h. Rats breathed spontaneously, and anesthesia was titrated to loss of righting reflex and tolerance of clip-style pulse oximetry. Six hours into the anesthetic, rats received 200 mg/kg bromodeoxyuridine (BrdU) intraperitoneally and were killed hours later. Frozen hippocampal sections were collected and processed for BrdU using an immunoperoxidase technique. BrdU(+) cells in the dentate gyrus were then counted, and compared with unanesthetized controls to determine the degree of new cell production. All four anesthetics were given to young rats. Older rats received isoflurane and ketamine, and also received isoflurane during their dark phase. RESULTS: Forty-two young, and 26 older, middle-aged rats were studied. When compared with controls, prolonged anesthesia in young rats with any drug had no effect on the number of BrdU(+) cells. BrdU labeling was also unaffected in older rats given isoflurane for 8 h during the light phase. Older rats had significantly lower BrdU(+) cell counts than younger rats. In older rats, ketamine anesthesia reduced BrdU(+) cell counts by 26% when compared with unanesthetized controls. Older rats given isoflurane for 8 h during their dark phase demonstrated no difference in BrdU labeling when compared with unanesthetized controls. CONCLUSION: Despite using multiple, mechanistically distinct drugs, we found no effect of prolonged anesthesia on adult hippocampal cell proliferation in young rats, a slight suppressive effect of ketamine in older rats, and no circadian effect with isoflurane. These data indicate that anesthetics are unlikely to alter cell proliferation, and by extension that anesthetic-induced inhibition of cell proliferation is unlikely to play a major role in postoperative cognitive impairment. The contrast between our findings, current concepts of anesthetic action, and known modifiers of cell proliferation suggest an incomplete understanding of the pharmacological and behavioral factors governing new neuron production.

Delayed suppression of hippocampal cell proliferation in rats following inescapable shocks.

Adult Sprague-Dawley rats were exposed to a single session of 100 inescapable tail shocks (IS). Bromodeoxyuridine (BrdU) was administered 1 h, 2 days or 7 days later and hippocampal cell proliferation (CP) was assessed after a 2-h survival period. Measures of plasma corticosterone (CORT) levels were also obtained. Despite a large increase in CORT immediately following IS, no associated change in CP was observed. In fact, the only significant change in CP was seen 7 days after IS, at a time when CORT was unchanged from control levels. These data raise questions about the general nature of the relationship between CORT and CP. They also suggest that, under some conditions, changes in hippocampal CP may emerge only after an "incubation period".

Despite strong behavioral disruption, Delta9-tetrahydrocannabinol does not affect cell proliferation in the adult mouse dentate gyrus.

Marijuana is a widely abused illicit drug known to cause significant cognitive impairments. Marijuana has been hypothesized to target neurons in the hippocampus because of the abundance of cannabinoid receptors present in this structure. While there is no clear evidence of neuropathology in vivo, suppression of brain mitogenesis, and ultimately neurogenesis, may provide a sensitive index of marijuana's more subtle effects on neural mechanisms subserving cognitive functions. We examined the effects of different doses and treatment regimens of Delta(9)-tetrahydrocannabinol (THC), the main active ingredient in marijuana, on cell proliferation in the dentate gyrus of adult male mice. Following drug treatment, the thymidine analog 5-bromo-2'-deoxyuridine (BrdU; 200 mg/kg, i.p.) was administered two hours prior to sacrifice to assess cell proliferation, the first step in neurogenesis. Administration of THC produced dose-dependent catalepsy and suppression of motor activity. The number of BrdU-labeled cells was not significantly changed from vehicle control levels following either acute (1, 3, 10, 30 mg/kg, i.p.), sequential (two injections of 10 or 30 mg/kg, i.p., separated by 5 h), or chronic escalating (20 to 80 mg/kg, p.o.; for 3 weeks) drug administration. Furthermore, acute administration of the potent synthetic cannabinoid receptor agonist R-(+)-WIN 55,212-2 (WIN; 5 mg/kg, i.p.) also had no significant effect on cell proliferation. These findings provide no evidence for an effect of THC on hippocampal cell proliferation, even at doses producing gross behavioral intoxication. Whether marijuana or THC affects neurogenesis remains to be explored.

Circadian variation in mouse hippocampal cell proliferation.

Hippocampal cell proliferation and concomitant motor activity were examined in adult male mice (C57BL/6J) across a 12:12h light-dark cycle. 5-Bromo-2'-deoxyuridine (BrdU) (200 mg/kg, i.p.) was administered at six equally spaced time points across 24h. A significant change in cell proliferation was found in the hilus (light phase>dark phase), but not in the granule cell layer (GCL)/subgranular zone (SGZ). Since it is generally believed that proliferating cells in the hilus and GCL/SGZ give rise primarily to glia and neurons, respectively, these data suggest a possible circadian influence on gliogenesis, rather than neurogenesis.

Repeated brief social defeat episodes in mice: effects on cell proliferation in the dentate gyrus.

Stressful experiences can affect hippocampal structure and function and can suppress new cell birth in the adult hippocampus in several species. Here we examine how repeated intermittent social defeat affects cell proliferation in the dentate gyrus (DG) in mice. Adult male CFW mice were subjected to 10 daily social defeat episodes, 3 defeat episodes within one day or a single defeat episode. Intruder mice were injected with 5-bromo-2'-deoxyuridine (BrdU, 200mg/kg, i.p.) 1h after the last fight, and incorporation of BrdU into proliferating cells in the DG was quantified. In a third experiment, aggressive resident mice were allowed to fight with an intruder mouse every day for 10 days, and these residents were injected with BrdU 1h after the last aggressive encounter. There was a significant decrease in cell proliferation in mice that received 10 social defeats, confirming and extending earlier results. This decrease is correlated with the intensity of the defeat experiences, as quantified by frequency of attack bites. Cell proliferation was slightly inhibited after a single defeat, although this effect was not significant. Three defeats within a 5-h period had no effect on levels of proliferation. Offensive aggressive stress in the residents did not result in any changes in hippocampal cell proliferation. These data indicate that repeated intermittent social defeat experienced over multiple days suppresses proliferation in the DG, and this may have important implications for our understanding of hippocampal changes related to stress psychopathologies.