The supplementation of a high dose of fish oil during pregnancy and lactation led to an elevation in Mfsd2a expression without any changes in docosahexaenoic acid levels in the retina of healthy 2-month-old mouse offspring.

Introduction: During fetal development, the proper development of neural and visual systems relies on the maternal supplementation of omega-3 fatty acids through placental transfer. Pregnant women are strongly advised to augment their diet with additional sources of omega-3, such as fish oil (FO). This supplementation has been linked to a reduced risk of preterm birth, pre-eclampsia, and perinatal depression. Recently, higher doses of omega-3 supplementation have been recommended for pregnant women. Considering that omega-3 fatty acids, particularly docosahexaenoic acid (DHA), play a crucial role in maintaining the delicate homeostasis required for the proper functioning of the retina and photoreceptors the effects of high-dose fish oil (FO) supplementation during pregnancy and lactation on the retina and retinal pigmented epithelium (RPE) in healthy offspring warrant better understanding. Methods: The fatty acid content and the changes in the expression of the genes regulating cholesterol homeostasis and DHA transport in the retina and RPE were evaluated following the high-dose FO supplementation. Results: Our study demonstrated that despite the high-dose FO treatment during pregnancy and lactation, the rigorous DHA homeostasis in the retina and RPE of the two-month-old offspring remained balanced. Another significant finding of this study is the increase in the expression levels of major facilitator superfamily domain-containing protein (Mfsd2a), a primary DHA transporter. Mfsd2a also serves as a major regulator of transcytosis during development, and a reduction in Mfsd2a levels poses a major risk for the development of leaky blood vessels. Conclusion: Impairment of the blood-retinal barrier (BRB) is associated with the development of numerous ocular diseases, and a better understanding of how to manipulate transcytosis in the BRB during development can enhance drug delivery through the BRB or contribute to the repair of central nervous system (CNS) barriers.

Amyloid-ß plaque formation and BACE1 accumulation in the brains of a 5xFAD Alzheimer's disease mouse model is associated with altered distribution and not proteolysis of BACE1 substrates Sez6 and Sez6L.

The formation of amyloid-ß peptides (Aß), that accumulate in Alzheimer's disease (AD) brains, involves proteolytic processing of the amyloid precursor protein (APP) firstly by ß-secretase (BACE1). Since BACE1 cleaves a plethora of other substrates, in this work we investigated whether the proteolysis and/or distribution of other BACE1 substrates, such as seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), is altered in AD. To test this we used 5xFAD mouse model brains that show an early accumulation of Aß plaques already at 2-months of age. Here we show for the first time that accumulation of BACE1 in peri-plaque regions and its enhanced levels in AD brains does not affect proteolysis of BACE1 substrates other than APP, such as Sez6 and Sez6L. We observed altered distribution of Sez6 and Sez6L in the area of Aß plaques in 5xFAD brains which is distinct to that of APP, BACE1 and/or LAMP1, suggesting different localization and/or function of these BACE1 substrates. While it is necessary to further elucidate the potential role that this may play in the course of AD, it is likely that Aß-targeted therapies may have beneficial effects against accumulation and/or altered distribution of BACE1 and its substrates, in addition to APP.

Calorie restriction changes the anxiety-like behaviour of ageing male Wistar rats in an onset- and duration-dependent manner.

Although initially recognized as a universally beneficial approach for the prevention of age-related impairments, the outcome of calorie restriction (CR) is now known to depend on several factors, most notably the age of the subject at the CR commencement, and CR duration. We aimed to examine if and how CR affects anxiety-like behaviour when it is introduced at middle age and late middle age. In addition, as the dopaminergic system is one of the main neurotransmitter systems involved in controlling anxiety, we examined the expression of dopamine receptors (D1R, D2R) in the cortex, striatum, and mesencephalon of male Wistar rats of varying ages. The study was performed on rats fed ad libitum (AL) or exposed to calorie restriction (60% of AL intake). Open field and light-dark tests were used to study anxiety-like behaviour, while PCR and Western blot were used to examine the expression of dopamine receptors. Calorie restriction implemented at middle-age led to variable outcomes on anxiety-like behaviour, while CR implemented at late middle age increased anxiety and decreased the availability of D2R levels in the cortex and mesencephalon. Taken together, these results advise caution when implementing calorie restriction late in life.

Late-Onset Calorie Restriction Worsens Cognitive Performances and Increases Frailty Level in Female Wistar Rats.

The current study aims to determine the potential benefits of calorie restriction (CR), one of the most promising paradigms for life span and healthspan extension, on cognitive performances in female Wistar rats during aging. As a measure of a healthspan, we evaluated the effects of different onset and duration of CR on frailty level. Female Wistar rats were exposed to either ad libitum (AL) or CR (60% of AL daily intake) food intake during aging. Two different CR protocols were used, life-long CR with an early-onset that started at the adult stage (6 months) and 3-month-long CR, started at the middle (15 months) and late-middle (21 months) age, thus defined as a late-onset CR. The effects of CR were evaluated using open-field, Y-maze, and novel object recognition tests. We broadened 2 tools for frailty assessment currently in use for experimental animals, and in alignment with our previous study, we created a physical-cognitive frailty tool that combines both physical and cognitive performances. Our results clearly showed that CR effects are highly dependent on CR duration and onset. While a life-long restriction with an early-onset has been proven as protective and beneficial, short-term restriction introduced at late age significantly worsens an animal's behavior and frailty. These results complement our previous study conducted in males and contribute to the understanding of sex differences in a response to CR during aging.

Neuroprotective effects of food restriction in a rat model of traumatic brain injury - the role of glucocorticoid signaling.

OBJECTIVE: Traumatic brain injury (TBI) is one of the most common causes of neurological damage in young and middle aged people. Food restriction (FR) has been shown to act neuroprotectively in animal models of stroke and TBI. Indeed, our previous studies showed that FR attenuates inflammation, through suppression of microglial activation and TNF-α production, suppresses caspase-3-induced neuronal cell death and enhances neuroplasticity in the rat model of TBI. Glucocorticoids (GCs) play a central role in mediating both molecular and behavioral responses to food restriction. However, the exact mechanisms of FR neuroprotection in TBI are still unclear. The goal of the present study was to examine whether FR exerts its beneficial effects by altering the glucocorticoid receptor (GR) signaling alone and/or together with other protective factors. METHODS: To this end, we examined the effects of FR (50% of regular daily food intake for 3 months prior to TBI) on the protein levels of total GR, GR phosphoisoform Ser232 (p-GR) and its transcriptional activity, as well as 11ß-HSD1, NFκB (p65) and HSP70 as factors related to the GR signaling. RESULTS: Our results demonstrate that FR applied prior to TBI significantly changes p-GR levels, and it's transcriptional activity during the recovery period after TBI. Moreover, as a pretreatment, FR modulates other protective factors in response to TBI, such as 11ß-HSD1, NF-κB (p65) and HSP70 that act in parallel with GR in it's anti-inflammatory and neuroprotective effects in the rat model of brain injury. CONCLUSION: Our results suggest that prophylactic FR represents a potent non-invasive approach capable of changing GR signalling, together with other factors related to the GR signaling in the model of TBI.

Corticosterone and Glucocorticoid Receptor in the Cortex of Rats during Aging-The Effects of Long-Term Food Restriction.

Numerous beneficial effects of food restriction on aging and age-related pathologies are well documented. It is also well-established that both short- and long-term food restriction regimens induce elevated circulating levels of glucocorticoids, stress-induced hormones produced by adrenal glands that can also exert deleterious effects on the brain. In the present study, we examined the effect of long-term food restriction on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the cortex during aging, in 18- and 24-month-old rats. Corticosterone level was increased in the cortex of aged ad libitum-fed rats. Food restriction induced its further increase, accompanied with an increase in the level of 11ß-hydroxysteroid dehydrogenase type 1. However, alterations in the level of GR phosphorylated at Ser232 were not detected in animals on food restriction, in line with unaltered CDK5 level, the decrease of Hsp90, and an increase in a negative regulator of GR function, FKBP51. Moreover, our data revealed that reduced food intake prevented age-related increase in the levels of NFκB, gfap, and bax, confirming its anti-inflammatory and anti-apoptotic effects. Along with an increase in the levels of c-fos, our study provides additional evidences that food restriction affects cortical responsiveness to glucocorticoids during aging.

Every-other-day feeding exacerbates inflammation and neuronal deficits in 5XFAD mouse model of Alzheimer's disease.

Food restriction has been widely associated with beneficial effects on brain aging and age-related neurodegenerative diseases such as Alzheimer's disease. However, previous studies on the effects of food restriction on aging- or pathology-related cognitive decline are controversial, emphasizing the importance of the type, onset and duration of food restriction. In the present study, we assessed the effects of preventive every-other-day (EOD) feeding regimen on neurodegenerative phenotype in 5XFAD transgenic mice, a commonly used mouse model of Alzheimer's disease. EOD feeding regimen was introduced to transgenic female mice at the age of 2 months and the effects on amyloid-ß (Aß) accumulation, gliosis, synaptic plasticity, and blood-brain barrier breakdown were analyzed in cortical tissue of 6-month-old animals. Surprisingly, significant increase of inflammation in the cortex of 5XFAD fed EOD mice was observed, reflected by the expression of microglial and astrocytic markers. This increase in reactivity and/or proliferation of glial cells was accompanied by an increase in proinflammatory cytokine TNF-α, p38 MAPK and EAAT2, and a decrease in GAD67. NMDA receptor subunit 2B, related to glutamate excitotoxicity, was increased in the cortex of 5XFAD-EOD mice indicating additional alterations in glutamatergic signaling. Furthermore, 4 months of EOD feeding regimen had led to synaptic plasticity proteins reduction and neuronal injury in 5XFAD mice. However, EOD feeding regimen did not affect Aß load and blood-brain barrier permeability in the cortex of 5XFAD mice. Our results demonstrate that EOD feeding regimen exacerbates Alzheimer's disease-like neurodegenerative and neuroinflammatory changes irrespective of Aß pathology in 5XFAD mice, suggesting that caution should be paid when using food restrictions in the prodromal phase of this neurodegenerative disease.

Short-term fish oil supplementation applied in presymptomatic stage of Alzheimer's disease enhances microglial/macrophage barrier and prevents neuritic dystrophy in parietal cortex of 5xFAD mouse model.

Dystrophic neurites and activated microglia are one of the main neuropathological characteristics of Alzheimer's disease (AD). Although the use of supplements with omega-3 fatty acids has been associated with reduced risk and lessened AD pathology, it still remains elusive whether such a treatment could affect dystrophic neurites (DNs) formation and microglia/macrophage behavior in the early phase of disease. We analyzed the effects of short-term (3 weeks) fish oil supplementation on DNs formation, tau hyperphosphorylation, Amyloid-beta peptide 1-42 (Aß42) levels and microglial/macrophage response to AD pathology in the parietal cortex of 4-month-old 5xFAD mice, a mouse model of AD. The present study shows for the first time that short-term FO supplementation applied in presymptomatic stage of AD, alters the behaviour of microglia/macrophages prompting them to establish a physical barrier around amyloid plaques. This barrier significantly suppresses DNs formation through the reduction of both Aß content and tau hyperphosphorylation. Moreover, the short-term FO treatment neither suppresses inflammation nor enhances phagocytic properties of microglia/macrophages in the response to Aß pathology, the effects most commonly attributed to the fish oil supplementation. Our findings suggest that fish oil consumption may play an important role in modulating microglial/macrophage response and ameliorating the AD pathology in presymptomatic stage of Alzheimer's disease.

Short-Term Fish Oil Treatment Changes the Composition of Phospholipids While Not Affecting the Expression of Mfsd2a Omega-3 Transporter in the Brain and Liver of the 5xFAD Mouse Model of Alzheimer's Disease.

Long-term fish oil (FO) supplementation is able to improve Alzheimer's disease (AD) pathology. We aimed to determine the impact of short-term fish oil (FO) intake on phospholipids composition and plaque pathology in 5xFAD mice, a widely used animal model of AD. A 3-week-long FO supplementation administered at 3 months of age decreased the number of dense core plaques in the 5xFAD cortex and changed phospholipids in the livers and brains of wild-type (Wt) and 5xFAD mice. Livers of both genotypes responded by increase of n-3 and reciprocal decrease of n-6 fatty acids. In Wt brains, FO supplementation induced elevation of n-3 fatty acids and subsequent enhancement of n-6/n-3 ratio. However, in 5xFAD brains the improved n-6/n-3 ratio was mainly due to FO-induced decrease in arachidonic and adrenic n-6 fatty acids. Also, brain and liver abundance of n-3 fatty acids were strongly correlated in Wts, oppositely to 5xFADs where significant brain-liver correlation exists only for n-6 fatty acids. Expression of omega-3 transporter Mfs2a remained unchanged after FO supplementation. We have demonstrated that even a short-term FO intake improves the phospholipid composition and has a significant effect on plaque burden in 5xFAD brains when applied in early stages of AD pathology.

The influence of propofol anesthesia exposure on nonaversive memory retrieval and expression of molecules involved in memory process in the dorsal hippocampus in peripubertal rats.

BACKGROUND: The effects of anesthetic drugs on postoperative cognitive function in children are not well defined and have not been experimentally addressed. AIMS: The present study aimed to examine the influence of propofol anesthesia exposure on nonaversive hippocampus-dependent learning and biochemical changes involved in memory process in the dorsal hippocampus, in peripubertal rats as the rodent model of periadolescence. METHODS: The intersession spatial habituation and the novel object recognition tasks were used to assess spatial and nonspatial, nonaversive hippocampus-dependent learning. The exposure to anesthesia was performed after comparably long acquisition phases in both tasks. Behavioral testing lasted for 2 consecutive days (24-hour retention period). Changes in the expression of molecules involved in memory retrieval/reconsolidation were examined in the dorsal hippocampus by Western blot and immunohistochemistry, at the time of behavioral testing. RESULTS: Exposure to propofol anesthesia resulted in inappropriate assessment of spatial novelty at the beginning of the test session and affected continuation of acquisition in the spatial habituation test. The treatment did not affect recognition of the novel object at the beginning of the test session but it attenuated overall preference to novelty, reflecting retrieval of a weak memory. The expression of phosphorylated extracellular signal-regulated kinase 2 (involved in memory retrieval) was decreased while the level of phosphorylated Ca2+ /calmodulin-dependent protein kinase IIα and early growth response protein 1 (involved in memory reconsolidation) was increased in the dorsal hippocampus. The level of Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog B (neuronal activity indicator) was increased in the dorsal dentate gyrus. Enhanced exploratory activity was still evident in the propofol anesthesia exposure (PAE) group 48 hour after the treatment in both tasks. CONCLUSION: In peripubertal rats, propofol anesthesia exposure affects memory retrieval and acquisition of new learning in the spatial and nonspatial, nonaversive learning tasks 24 hour after the treatment, along with the expression of molecules that participate in memory retrieval/reconsolidation in the dorsal hippocampus. These results may have clinical implications, favoring control of basic cognitive functions in older children after the propofol exposure.

Brain molecular changes and behavioral alterations induced by propofol anesthesia exposure in peripubertal rats.

BACKGROUND: Propofol is commonly used in modern anesthesiology. Some findings suggest that it is highly addictive. AIM: In this study it was examined whether propofol anesthesia exposure was able to induce behavioral alterations and brain molecular changes already described in addictive drug usage in peripubertal rats, during the onset of mid/periadolescence as a developmental period with increasing vulnerability to drug addiction. METHODS: The expression of D1 dopamine receptor, a dopamine, and cAMP-regulated phosphoprotein with a Mr 32 000; Ca2+ /calmodulin-dependent protein kinase IIα; and Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog-B was examined in peripubertal rats 4, 24, and 48 hour after propofol anesthesia exposure by Western blot and immunohistochemistry. Brain regions of interest were the medial prefrontal cortex, the striatum, and the thalamus. Anxiety and behavioral cross-sensitization to d-amphetamine were examined as well. RESULTS: Significant increase in the expression of dopamine and cAMP-regulated phosphoprotein with a Mr 32 000 phosphorylated at threonine 34, a postsynaptic marker of dopaminergic neurotransmission, and Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog-B, a marker of neuronal activity, was detected in the thalamus of experimental animals 4-24 hour after the treatment, with the accent on the paraventricular thalamic nucleus. Significant increase in the expression of Ca2+ /calmodulin-dependent protein kinase IIα phosphorylated at threonine 286, a sensor of synaptic activity, was observed in the prefrontal cortex and the striatum 24 hour after propofol anesthesia exposure. It was accompanied by a significant decrease in Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog-B expression in the striatum. Decreased behavioral inhibition in aversive environment and increased motor response to d-amphetamine in a context-independent manner were observed as well. CONCLUSION: In peripubertal rats, propofol anesthesia exposure induces transient molecular and behavioral response that share similarities with those reported previously for addictive drugs. In the absence of additional pharmacological manipulation, all detected effects receded within 48 hour after the treatment.

Neonatal Propofol Anesthesia Changes Expression of Synaptic Plasticity Proteins and Increases Stereotypic and Anxyolitic Behavior in Adult Rats.

Propofol is a general anesthetic commonly used in pediatric clinical practices. Experimental findings demonstrate that anesthetics induce widespread apoptosis and cognitive decline in a developing brain. Although anesthesia-mediated neurotoxicity is the most prominent during intense period of synaptogenesis, the effects of an early anesthesia exposure on the synapses are not well understood. The aim of this study was to examine the effects of neonatal propofol anesthesia on the expression of key proteins that participate in synaptogenesis and synaptic plasticity and to evaluate long-term neurobehavioral abnormalities in the mature adult brain. Propofol-injected 7-day-old rats were maintained under 2-, 4-, and 6-h-long anesthesia and sacrificed 0, 4, 16, and 24 h after the termination of each exposure. We showed that propofol anesthesia strongly influenced spatiotemporal expression and/or proteolytic processing of crucial presynaptic (GAP-43, synaptophysin, α-synuclein), trans-synaptic (N-cadherin), and postsynaptic (drebrin, MAP-2) proteins in the cortex and thalamus. An overall decrease of synaptophysin, α-synuclein, N-cadherin, and drebrin indicated impaired function and structure of the synaptic contacts immediately after anesthesia cessation. GAP-43 and MAP-2 adult and juvenile isoforms are upregulated following anesthesia, suggesting compensatory mechanism in the maintaining of the structural integrity and stabilization of developing axons and dendritic arbors. Neonatal propofol exposure significantly altered spontaneous motor activity (increased stereotypic/repetitive movements) and changed emotional behavior (reduced anxiety-like response) in the adulthood, 6 months later. These findings suggest that propofol anesthesia is synaptotoxic in the developing brain, disturbing synaptic dynamics and producing neuroplastic changes permanently incorporated into existing networks with long-lasting functional consequences.

The Fas Ligand/Fas Death Receptor Pathways Contribute to Propofol-Induced Apoptosis and Neuroinflammation in the Brain of Neonatal Rats.

A number of experimental studies have reported that exposure to common, clinically used anesthetics induce extensive neuroapoptosis and cognitive impairment when applied to young rodents, up to 2 weeks old, in phase of rapid synaptogenesis. Propofol is the most used general anesthetic in clinical practice whose mechanisms of neurotoxicity on the developing brain remains to be examined in depth. This study investigated effects of different exposures to propofol anesthesia on Fas receptor and Fas ligand expressions, which mediate proapoptotic and proinflammation signaling in the brain. Propofol (20 mg/kg) was administered to 7-day-old rats in multiple doses sufficient to maintain 2-, 4- and 6-h duration of anesthesia. Animals were sacrificed at 0, 4, 16 and 24 h after termination of anesthesia. It was found that propofol anesthesia induced Fas/FasL and downstream caspase-8 expression more prominently in the thalamus than in the cortex. Opposite, Bcl-2 and caspase-9, markers of intrinsic pathway activation, were shown to be more influenced by propofol treatment in the cortex. Further, we have established upregulation of caspase-1 and IL-1ß cytokine transcription as well as subsequent activation of microglia that is potentially associated with brain inflammation. Behavioral analyses revealed that P35 and P60 animals, neonatally exposed to propofol, had significantly higher motor activity during three consecutive days of testing in the open field, though formation of the intersession habituation was not prevented. This data, together with our previous results, contributes to elucidation of complex mechanisms of propofol toxicity in developing brain.

General Anesthesia Causes Epigenetic Histone Modulation of c-Fos and Brain-derived Neurotrophic Factor, Target Genes Important for Neuronal Development in the Immature Rat Hippocampus.

BACKGROUND: Early postnatal exposure to general anesthesia (GA) may be detrimental to brain development, resulting in long-term cognitive impairments. Older literature suggests that in utero exposure of rodents to GA causes cognitive impairments in the first-generation as well as in the second-generation offspring never exposed to GA. Thus, the authors hypothesize that transient exposure to GA during critical stages of synaptogenesis causes epigenetic changes in chromatin with deleterious effects on transcription of target genes crucial for proper synapse formation and cognitive development. They focus on the effects of GA on histone acetyltransferase activity of cAMP-responsive element-binding protein and the histone-3 acetylation status in the promoters of the target genes brain-derived neurotrophic factor and cellular Finkel-Biskis-Jinkins murine sarcoma virus osteosarcoma oncogene (c-Fos) known to regulate the development of neuronal morphology and function. METHODS: Seven-day-old rat pups were exposed to a sedative dose of midazolam followed by combined nitrous oxide and isoflurane anesthesia for 6 h. Hippocampal neurons and organotypic hippocampal slices were cultured in vitro and exposed to GA for 24 h. RESULTS: GA caused epigenetic modulations manifested as histone-3 hypoacetylation (decrease of 25 to 30%, n = 7 to 9) and fragmentation of cAMP-responsive element-binding protein (two-fold increase, n = 6) with 25% decrease in its histone acetyltransferase activity, which resulted in down-regulated transcription of brain-derived neurotrophic factor (0.2- to 0.4-fold, n = 7 to 8) and cellular Finkel-Biskis-Jinkins murine sarcoma virus osteosarcoma oncogene (about 0.2-fold, n = 10 to 12). Reversal of histone hypoacetylation with sodium butyrate blocked GA-induced morphological and functional impairments of neuronal development and synaptic communication. CONCLUSION: Long-term impairments of neuronal development and synaptic communication could be caused by GA-induced epigenetic phenomena.

Dietary restriction suppresses apoptotic cell death, promotes Bcl-2 and Bcl-xl mRNA expression and increases the Bcl-2/Bax protein ratio in the rat cortex after cortical injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability in humans. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. Using an animal model of TBI we examined the effect of dietary restriction (DR) on the neuroapoptosis and Bcl-2 family genes as the main regulators of the intrinsic apoptotic pathway. Bcl-2, Bcl-xl and Bax mRNA and protein expression in the ipsilateral cortex of adult Wistar rats exposed to DR before TBI were studied from 2 to 28 days post injury. Our results showed that DR suppressed neuroapoptosis and promoted significant upregulation of antiapoptotic Bcl-2 and Bcl-xl mRNAs in the ipsilateral cortex following injury. Expression of the proapoptotic Bax gene increased in ad libitum (AL) fed rats but remained unchanged in rats exposed to DR. Although the expression of Bcl-2, Bcl-xl and Bax proteins was changed in a similar manner in both experimental groups, DR promoted a continuous increase in the Bcl-2:Bax protein ratio throughout the recovery period. Together with our previous finding that DR mediates inhibition of the extrinsic apoptotic pathway the present work reveals that modulation of the intrinsic pathway contributes to the beneficial effect of DR in brain injury. These findings provide new insight into the effects of DR on pro-survival signaling after injury, lending further support to its neuroprotective effect.

Neonatal propofol anesthesia modifies activity-dependent processes and induces transient hyperlocomotor response to d-amphetamine during adolescence in rats.

This study examined the influence of propofol anesthesia on the expression of activity-regulated molecules (BDNF and c-Fos) and synaptic plasticity markers (synaptophysin, GAP-43, drebrin) in the frontal cortex and thalamus of 7-day-old (P7) rats. Although these brain regions are the main targets of anesthetic action, they are contained in the cortico-striato-thalamo-cortical feedback loops, involved in naturally occurring and drug-induced psychoses. Therefore, functional integrity of these loops was examined in adolescent and adult rats through d-amphetamine-induced hyperactivity. Propofol treatment (25mg/kg) decreased exon-specific and total BDNF mRNA expression in the frontal cortex and thalamus, in a time-dependent manner. BDNF protein level was increased in the frontal cortex and decreased in the thalamus, which was accompanied by the change of phospho-TrkB expression. Similarly to BDNF, the expression of c-Fos was decreased in the frontal cortex while it was changed only at the protein level in the thalamus. Synaptic plasticity markers changed in a time- and region-specific manner, indicating increased synaptogenesis in the frontal cortex and synapse elimination in the thalamus in P7 rats after the propofol anesthesia exposure. These early molecular changes were followed by time-related, increased motor reaction to d-amphetamine in adolescent, but not in adult rats. Our study revealed that exposure of immature brain to propofol anesthesia during the critical phase of development provoked immediate changes in activity-dependent processes and synaptic adjustment, influencing brain capacity to integrate later developmental events and resulting in temporary altered response to acute psychotropic stimulation during adolescence.

Induction of TNF-α signaling cascade in neonatal rat brain during propofol anesthesia.

Propofol anesthesia can trigger pro- and anti-apoptotic signaling pathways in the rat brain. In our previous work, we demonstrated that propofol causes widespread apoptotic neurodegeneration in 7-postnatal-day-old (PND7) but not in PND14 rat neurons. The mechanism responsible for these opposing outcomes is unknown, apparently linked to the specific stage of brain development. The present study aims to elucidate the anti-apoptotic process that is activated in the cortex and thalamus of PND14 Wistar rats during the first 48 h after the onset of propofol anesthesia. We showed that the expression of tumor necrosis factor-α (TNF-α) and several components of its pathway, TNFR1 and caspase-8, was significantly increased in the cortex and thalamus. Nuclear factor kappa B (NF-κB) p65 was downregulated in the cortex and upregulated in the thalamus. The expression of c-Fos was upregulated only in the cortex, showing opposed profile compared to NF-κB p65. Double immunofluorescence staining revealed the colocalization of NF-κB p65 with neuronal marker (NeuN), but with predominantly cytoplasmic localization. Finally, X-linked inhibitor of apoptosis protein (XIAP) was upregulated in both examined structures. Immunohistochemical staining with Iba-1 revealed that the treatment did not induce changes in microglial morphology. Our results (i) reveal that the simultaneous activation of pro- and anti-apoptotic signaling occurs after propofol anesthesia, and (ii) pinpoint the potential neuroprotective role of XIAP in anesthesia-induced neurotoxicity.

Propofol anesthesia induces proapoptotic tumor necrosis factor-α and pro-nerve growth factor signaling and prosurvival Akt and XIAP expression in neonatal rat brain.

Previously we observed that prolonged exposure to propofol anesthesia causes caspase-3- and calpain-mediated neuronal death in the developing brain. The present study examines the effects of propofol anesthesia on the expression of tumor necrosis factor-α (TNFα), pro-nerve growth factor (NGF), and their receptors in the cortex and the thalamus. We also investigated how propofol influences the expression of Akt and X-linked inhibitor of apoptosis (XIAP) expression, proteins that promote prosurvival pathways. Seven-day-old rats (P7) were exposed to propofol anesthesia lasting 2, 4, or 6 hr and killed 0, 4, 16, or 24 hr after anesthesia termination. The relative levels of mRNA and protein expression were estimated by RT-PCR and Western blot analysis, respectively. The treatments caused marked activation of TNFα and its receptor TNFR-1 and pro-NGF and p75(NTR) receptor expression. In parallel with the induction of these prodeath signals, we established that propofol anesthesia promotes increased expression of the prosurvival molecules pAkt and XIAP during the 24-hr postanesthesia period. These results show that different brain structures respond to propofol anesthesia with a time- and duration of exposure-dependent increase in proapoptotic signaling and with concomitant increases in activities of prosurvival proteins. We hypothesized that the fine balance between these opposing processes sustains homeostasis in the immature rat brain and prevents unnecessary damage after exposure to an injurious stimulus. The existence of this highly regulated process provides a time frame for potential therapeutic intervention directed toward suppressing the deleterious component of propofol anesthesia.

Early exposure to general anesthesia disturbs mitochondrial fission and fusion in the developing rat brain.

BACKGROUND: General anesthetics induce apoptotic neurodegeneration in the developing mammalian brain. General anesthesia (GA) also causes significant disturbances in mitochondrial morphogenesis during intense synaptogenesis. Mitochondria are dynamic organelles that undergo remodeling via fusion and fission. The fine balance between these two opposing processes determines mitochondrial morphometric properties, allowing for their regeneration and enabling normal functioning. As mitochondria are exquisitely sensitive to anesthesia-induced damage, we examined how GA affects mitochondrial fusion/fission. METHODS: Seven-day-old rat pups received anesthesia containing a sedative dose of midazolam followed by a combined nitrous oxide and isoflurane anesthesia for 6 h. RESULTS: GA causes 30% upregulation of reactive oxygen species (n = 3-5 pups/group), accompanied by a 2-fold downregulation of an important scavenging enzyme, superoxide dismutase (n = 6 pups/group). Reactive oxygen species upregulation is associated with impaired mitochondrial fission/fusion balance, leading to excessive mitochondrial fission. The imbalance between fission and fusion is due to acute sequestration of the main fission protein, dynamin-related protein 1, from the cytoplasm to mitochondria, and its oligomerization on the outer mitochondrial membrane. These are necessary steps in the formation of the ring-like structures that are required for mitochondrial fission. The fission is further promoted by GA-induced 40% downregulation of cytosolic mitofusin-2, a protein necessary for maintaining the opposing process, mitochondrial fusion (n = 6 pups/group). CONCLUSIONS: Early exposure to GA causes acute reactive oxygen species upregulation and disturbs the fine balance between mitochondrial fission and fusion, leading to excessive fission and disturbed mitochondrial morphogenesis. These effects may play a causal role in GA-induced developmental neuroapoptosis.

Caloric restriction suppresses microglial activation and prevents neuroapoptosis following cortical injury in rats.

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI.