Maternal environmental enrichment protects neonatal brains from hypoxic-ischemic challenge by mitigating brain energetic dysfunction and modulating glial cell responses.

There is evidence that maternal milieu and changes in environmental factors during the prenatal period may exert a lasting impact on the brain health of the newborn, even in case of neonatal brain hypoxia-ischemia (HI). The present study aimed to investigate the effects of maternal environmental enrichment (EE) on HI-induced energetic and metabolic failure, along with subsequent neural cell responses in the early postnatal period. Male Wistar pups born to dams exposed to maternal EE or standard conditions (SC) were randomly divided into Sham-SC, HI-SC, Sham-EE, and HI-EE groups. Neonatal HI was induced on postnatal day (PND) 3. The Na+,K+-ATPase activity, mitochondrial function and neuroinflammatory related-proteins were assessed at 24 h and 48 h after HI. MicroPET-FDG scans were used to measure glucose uptake at three time points: 24 h post-HI, PND18, and PND24. Moreover, neuronal preservation and glial cell responses were evaluated at PND18. After HI, animals exposed to maternal EE showed an increase in Na+,K+-ATPase activity, preservation of mitochondrial potential/mass ratio, and a reduction in mitochondrial swelling. Glucose uptake was preserved in HI-EE animals from PND18 onwards. Maternal EE attenuated HI-induced cell degeneration, white matter injury, and reduced astrocyte immunofluorescence. Moreover, the HI-EE group exhibited elevated levels of IL-10 and a reduction in Iba-1 positive cells. Data suggested that the regulation of AKT/ERK1/2 signaling pathways could be involved in the effects of maternal EE. This study evidenced that antenatal environmental stimuli could promote bioenergetic and neural resilience in the offspring against early HI damage, supporting the translational value of pregnancy-focused environmental treatments.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain.

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

Experimental cerebral palsy causes microstructural brain damage in areas associated to motor deficits but no spatial memory impairments in the developing rat.

INTRODUCTION: Cerebral palsy (CP) is the major cause of motor and cognitive impairments during childhood. CP can result from direct or indirect structural injury to the developing brain. In this study, we aimed to describe brain damage and behavioural alterations during early adult life in a CP model using the combination of maternal inflammation, perinatal anoxia and postnatal sensorimotor restriction. METHODS: Pregnant Wistar rats were injected intraperitoneally with 200 µg/kg LPS at embryonic days E18 and E19. Between 3 and 6 h after birth (postnatal day 0 - PND0), pups of both sexes were exposed to anoxia for 20 min. From postnatal day 2 to 21, hindlimbs of animals were immobilized for 16 h daily during their active phase. From PND40, locomotor and cognitive tests were performed using Rota-Rod, Ladder Walking and Morris water Maze. Ex-vivo MRI Diffusion Tensor Imaging (DTI) and Neurite Orientation Dispersion and Density Imaging (NODDI) were used to assess macro and microstructural damage and brain volume alterations induced by the model. Myelination and expression of neuronal, astroglial and microglial markers, as well as apoptotic cell death were evaluated by immunofluorescence. RESULTS: CP animals showed decreased body weight, deficits in gross (rota-rod) and fine (ladder walking) motor tasks compared to Controls. No cognitive impairments were observed. Ex-vivo MRI showed decreased brain volumes and impaired microstructure in the cingulate gyrus and sensory cortex in CP brains. Histological analysis showed increased cell death, astrocytic reactivity and decreased thickness of the corpus callosum and altered myelination in CP animals. Hindlimb primary motor cortex analysis showed increased apoptosis in CP animals. Despite the increase in NeuN and GFAP, no differences between groups were observed as well as no co-localization with the apoptotic marker. However, an increase in Iba-1+ microglia with co-localization to cleaved caspase 3 was observed. CONCLUSION: Our results suggest that experimental CP induces long-term brain microstructural alterations in myelinated structures, cell death in the hindlimb primary motor cortex and locomotor impairments. Such new evidence of brain damage could help to better understand CP pathophysiological mechanisms and guide further research for neuroprotective and neurorehabilitative strategies for CP patients.

Pregnancy swimming prevents early brain mitochondrial dysfunction and causes sex-related long-term neuroprotection following neonatal hypoxia-ischemia in rats.

Neonatal hypoxia-ischemia (HI) is a major cause of cognitive impairments in infants. Antenatal strategies improving the intrauterine environment can have high impact decreasing pregnancy-derived intercurrences. Physical exercise alters the mother-fetus unity and has been shown to prevent the energetic challenge imposed by HI. This study aimed to reveal neuroprotective mechanisms afforded by pregnancy swimming on early metabolic failure and late cognitive damage, considering animals' sex as a variable. Pregnant Wistar rats were submitted to daily swimming exercise (20' in a tank filled with 32 °C water) during pregnancy. Neonatal HI was performed in male and female pups at postnatal day 7. Electron chain transport, mitochondrial mass and function and ROS formation were assessed in the right brain hemisphere 24 h after HI. From PND45, reference and working spatial memory were tested in the Morris water maze. MicroPET-FDG images were acquired 24 h after injury (PND8) and at PND60, following behavioral analysis. HI induced early energetic failure, decreased enzymatic activity in electron transport chain, increased production of ROS in cortex and hippocampus as well as caused brain glucose metabolism dysfunction and late cognitive impairments. Maternal swimming was able to prevent mitochondrial dysfunction and to improve spatial memory. The intergenerational effects of swimming were sex-specific, since male rats were benefited most. In conclusion, maternal swimming was able to affect the mitochondrial response to HI in the offspring's brains, preserving its function and preventing cognitive damage in a sex-dependent manner, adding relevant information on maternal exercise neuroprotection and highlighting the importance of mitochondria as a therapeutic target for HI neuropathology.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

Early-life stress affects behavioral and neurochemical parameters differently in male and female juvenile Wistar rats.

Neonatal handling is an early life stressor that leads to behavioral and neurochemical changes in adult rats in a sex-specific manner and possibly affects earlier stages of development. Here, we investigated the effects of neonatal handling (days 1-10 after birth) on juvenile rats focusing on biochemical parameters and olfactory memory after weaning. Male neonatal handled rats performed more crossings on the hole-board task, increased Na+ /K+ -ATPase activity in the olfactory bulb, and decreased acetylcholinesterase activity in the hippocampus versus non-handled males. Female neonatal handled animals increased the number of rearing and nose-pokes on the hole-board task, decreased glutathione peroxidase activity, and total thiol content in the hippocampus versus non-handled females. This study reinforces that early life stress affects behavioral and neurochemical parameters in a sex-specific manner even before the puberty onset.

Chronic mild hyperhomocysteinemia induces anxiety-like symptoms, aversive memory deficits and hippocampus atrophy in adult rats: New insights into physiopathological mechanisms.

In the last decade, increased homocysteine levels have been implicated as a risk factor for neurodegenerative and psychiatric disorders. We have developed an experimental model of chronic mild hyperhomocysteinemia (HHcy) in order to observe metabolic impairments in the brain of adult rodents. Besides its known effects on brain metabolism, the present study sought to investigate whether chronic mild HHcy could induce learning/memory impairments associated with biochemical and histological damage to the hippocampus. Adult male Wistar rats received daily subcutaneous injections of homocysteine (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th day of life or saline solution (Controls). After injections, anxiety-like and memory tests were performed. Following behavioral analyses, brains were sliced and hippocampal volumes assessed and homogenized for redox state assessment, antioxidant activity, mitochondrial functioning (chain respiratory enzymes and ATP levels) and DNA damage analyses. Behavioral analyses showed that chronic mild HHcy may induce anxiety-like behavior and impair long-term aversive memory (24 h) that was evaluated by inhibitory avoidance task. Mild HHcy decreased locomotor and/or exploratory activities in elevated plus maze test and caused hippocampal atrophy. Decrease in cytochrome c oxidase, DNA damage and redox state changes were also observed in hippocampus of adult rats subjected to mild HHcy. Our findings show that chronic mild HHcy alters biochemical and histological parameters in the hippocampus, leading to behavioral impairments. These findings might be considered in future studies aiming to search for alternative strategies for treating the behavioral impairments in patients with mild elevations in homocysteine levels.

Arundic acid administration protects astrocytes, recovers histological damage and memory deficits induced by neonatal hypoxia ischemia in rats.

INTRODUCTION: Perinatal hypoxia-ischemia (HI) is one of the main causes of mortality and chronic neurological morbidity in infants and children. Astrocytes play a key role in HI progression, becoming reactive in response to the injury, releasing S100 calcium binding protein B (S100B). Since S100B inhibition seems to have neuroprotective effects on central nervous system injury models, here we evaluated the neuroprotective effects of an S100B inhibitor, arundic acid (AA) in a HI model. METHODS: On the 7th postnatal day, animals were submitted to the combination of common carotid artery occlusion and hypoxic atmosphere (8% O2) for 60 min. Three experiments were performed in order to: (1) define AA dose (0.1, 1 or 10 mg/kg, pre-hypoxia i.p. injection), (2) test if repeated AA administrations (10 mg/kg at 3 time points: Pre-hypoxia, 24 h and 48 h after HI) would improve the response and (3) investigate biochemical mechanisms involved in AA protection two days after HI. RESULTS: AA at a dose of 10 mg/kg applied before and after hypoxia, was the only treatment protocol that was able to improve HI-induced memory deficits, to reduce tissue damage, to promote astrocytic survival in the hippocampus and to reduced extracellular release of S100B in the cerebrospinal fluid. CONCLUSION: Overall, AA treatment showed beneficial effects on memory deficits, tissue damage, promoting astrocyte survival likely by reducing S100B release. Protection aided to astrocytes by AA treatment against HI lesion may lead to development of new therapeutic strategies that target these particular cells.

Effect of Sciatic Nerve Transection on acetylcholinesterase activity in spinal cord and skeletal muscles of the bullfrog Lithobates catesbeianus / Efeito da Secção do Nervo Isquiático sobre a atividade da acetilcolinesterase em medula espinal e músculos esqueléticos da rã-touro Lithobates catesbeianus

Abstract Sciatic nerve transection (SNT), a model for studying neuropathic pain, mimics the clinical symptoms of "phantom limb", a pain condition that arises in humans after amputation or transverse spinal lesions. In some vertebrate tissues, this condition decreases acetylcholinesterase (AChE) activity, the enzyme responsible for fast hydrolysis of released acetylcholine in cholinergic synapses. In spinal cord of frog Rana pipiens, this enzyme's activity was not significantly changed in the first days following ventral root transection, another model for studying neuropathic pain. An answerable question is whether SNT decreases AChE activity in spinal cord of frog Lithobates catesbeianus, a species that has been used as a model for studying SNT-induced neuropathic pain. Since each animal model has been created with a specific methodology, and the findings tend to vary widely with slight changes in the method used to induce pain, our study assessed AChE activity 3 and 10 days after complete SNT in lumbosacral spinal cord of adult male bullfrog Lithobates catesbeianus. Because there are time scale differences of motor endplate maturation in rat skeletal muscles, our study also measured the AChE activity in bullfrog tibial posticus (a postural muscle) and gastrocnemius (a typical skeletal muscle that is frequently used to study the motor system) muscles. AChE activity did not show significant changes 3 and 10 days following SNT in spinal cord. Also, no significant change occurred in AChE activity in tibial posticus and gastrocnemius muscles at day 3. However, a significant decrease was found at day 10, with reductions of 18% and 20% in tibial posticus and gastrocnemius, respectively. At present we cannot explain this change in AChE activity. While temporally different, the direction of the change was similar to that described for rats. This similarity indicates that bullfrog is a valid model for investigating AChE activity following SNT.

Resumo A transecção do nervo isquiático (SNT), um modelo para estudar dor neuropática, simula os sintomas clínicos do "membro fantasma", uma condição dolorosa que ocorre nos humanos após amputação ou secção completa da medula espinal. Essa condição muda a atividade da acetilcolinesterase (AChE), a enzima responsável pela rápida hidrólise da acetilcolina liberada nas sinapses colinérgicas, em alguns tecidos de vertebrados. Em medula espinal de rã Rana pipiens, a atividade da AChE não foi significativamente alterada nos primeiros dias após a secção da raiz ventral, outro modelo para o estudo da dor neuropática. Uma questão ainda não respondida é se a SNT diminui a atividade da AChE na medula espinal de rã Lithobates catesbeianus, uma espécie que vem sendo usada como modelo em estudos da dor neuropática induzida por SNT. Como cada modelo animal é criado a partir de metodologia específica, e seus resultados tendem a variar com pequenas mudanças na metodologia de indução da dor, o presente estudo avaliou a atividade da AChE em medula espinal lombossacral de rã-touro Lithobates catesbeianus, adultos, machos, 3 e 10 dias após a completa SNT. Como há diferenças temporais na maturação de placas motoras em músculos esqueléticos de ratos, nosso estudo ainda demonstrou, na rã-touro, os efeitos da SNT sobre a atividade da AChE nos músculos esqueléticos tibial posticus, um músculo postural, e gastrocnêmio, um músculo frequentemente usado em estudos do sistema motor. A atividade da AChE não mudou significativamente na medula espinal aos 3 e 10 dias após a SNT. Nos músculos, a atividade não alterou significativamente aos 3 dias após a lesão, mas reduziu de forma significativa aos 10 dias após a SNT. Aos 10 dias, a diminuição foi 18% no músculo tibial posticus e 20% no gastrocnêmio. No momento, nós não temos explicação para essa mudança na atividade da AChE. Embora temporalmente diferente, o sentido da mudança é similar ao que é descrito em ratos. Esta similaridade torna a rã-touro um modelo válido para se estudar questões ainda não respondidas da SNT sobre a AChE.

Glial glutamate transporters expression, glutamate uptake, and oxidative stress in an experimental rat model of intracerebral hemorrhage.

Glial glutamate transporters (EAAT1 and EAAT2), glutamate uptake, and oxidative stress are important players in the pathogenesis of ischemic brain injury. However, the changes in EAAT1 and EAAT2 expression, glutamate uptake and the oxidative profile during intracerebral hemorrhage (ICH) development have not been described. The present study sought to investigate the changes of the above-mentioned variables, as well as the Na+/K+-ATPase and glutamine synthetase activities (as important contributors of glutamate homeostasis) and the percentage of neuronal cells after 6 h, 24 h, 72 h and 7 days of ICH. An injection of 0.2U of bacterial collagenase in the ipsilateral striatum was used to induce ICH in male Wistar rats; naïve animals were used as controls. EAAT1 and EAAT2 expression and glutamate uptake in the ipsilateral striatum were assessed. Additionally, the percentage of MAP2+ cells, Na+/K+-ATPase and GS activities, as well as the oxidative profile were analyzed. It is shown a decrease of EAAT1 expression and glutamate uptake 6 h post-ICH, whereas EAAT2 decreased 72 h after the event; conversely EAAT2 and glutamate uptake were increased after 7 days. The oxidative stress and endogenous defense system exhibited a remarkable response at 72 h of injury. ICH also increased Na+/K+-ATPase activity and selectively decreased GS activity, variables known to be important contributors of glial glutamate transporters activities. Altogether, present findings indicate that ICH induces different temporal EAAT1 and EAAT2 responses, culminating with an imbalance of glutamate uptake capacity, increased oxidative stress and sustained neuronal loss.

Effect of Sciatic Nerve Transection on acetylcholinesterase activity in spinal cord and skeletal muscles of the bullfrog Lithobates catesbeianus.

Sciatic nerve transection (SNT), a model for studying neuropathic pain, mimics the clinical symptoms of "phantom limb", a pain condition that arises in humans after amputation or transverse spinal lesions. In some vertebrate tissues, this condition decreases acetylcholinesterase (AChE) activity, the enzyme responsible for fast hydrolysis of released acetylcholine in cholinergic synapses. In spinal cord of frog Rana pipiens, this enzyme's activity was not significantly changed in the first days following ventral root transection, another model for studying neuropathic pain. An answerable question is whether SNT decreases AChE activity in spinal cord of frog Lithobates catesbeianus, a species that has been used as a model for studying SNT-induced neuropathic pain. Since each animal model has been created with a specific methodology, and the findings tend to vary widely with slight changes in the method used to induce pain, our study assessed AChE activity 3 and 10 days after complete SNT in lumbosacral spinal cord of adult male bullfrog Lithobates catesbeianus. Because there are time scale differences of motor endplate maturation in rat skeletal muscles, our study also measured the AChE activity in bullfrog tibial posticus (a postural muscle) and gastrocnemius (a typical skeletal muscle that is frequently used to study the motor system) muscles. AChE activity did not show significant changes 3 and 10 days following SNT in spinal cord. Also, no significant change occurred in AChE activity in tibial posticus and gastrocnemius muscles at day 3. However, a significant decrease was found at day 10, with reductions of 18% and 20% in tibial posticus and gastrocnemius, respectively. At present we cannot explain this change in AChE activity. While temporally different, the direction of the change was similar to that described for rats. This similarity indicates that bullfrog is a valid model for investigating AChE activity following SNT.

Galantamine administration reduces reactive astrogliosis and upregulates the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia.

Neonatal hypoxia ischemia (HI) plays a role in the etiology of several neurological pathologies and causes severe sequelae. Acetylcholine is a neurotransmitter in the central nervous system and cholinesterase inhibitors have demonstrated a positive action over HI induced deficits. In order to evaluate the effects of pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in a model of perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right carotid artery with the exposure to a 1h hypoxia. Intraperitoneal injections of galantamine were administered in two different protocols: one pre and other post-hypoxia. The analysis of brain structures volume at PND45 showed that pre-hypoxia galantamine treatment prevented tissue injury to the ipsilesional hippocampus. Also, immunofluorescence showed HI-induced increase in the number of astrocytes that was prevented by pre-hypoxia treatment. Biochemical analysis was performed in the ipsilesional hippocampus at PND8 and revealed that pre-hypoxia galantamine treatment: 1) prevented the neuronal loss induced by HI; 2) reduced the HI-induced hypertrophy of astrocytes; and 3) caused an increase in the activity of the anti-oxidant enzyme catalase. Overall, treatment with galantamine was able to prevent the brain damage, increase the survival of neurons, reduce astrocytic reaction and increase the activity of the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia.

The use of cold coagulation for the treatment of cervical intraepithelial neoplasia.

In 2015, Cold Coagulation was introduced as a treatment for cervical intraepithelial neoplasia (CIN) at our colposcopy clinic. We reviewed the 6-month follow up data of the first 200 women who underwent Cold Coagulation using cytology and HPV status as tests of cure (TOC). A random sample of 200 patients treated by Large Loop Excision of the Transformation Zone (LLETZ) during the same period was used to compare treatment outcome. Six months following treatment,173 (86.5%) of the women treated by CC and 167 (83.5%) treated by LLETZ had negative cytology. (x2= P>0.05). 148 (74%) treated by Cold Coagulation and 166 (83%) treated by LLETZ were HPV negative (x2= P<0.05). One hundred and thirty-nine (70%) women treated by Cold Coagulation and 152 (76%) treated with LLETZ had normal cytology and were HPV negative. This audit of our initial experience supports the observation that Cold Coagulation is as effective as LLETZ in the management of CIN when cervical cytology is used as a test of cure.

Administration of Huperzia quadrifariata Extract, a Cholinesterase Inhibitory Alkaloid Mixture, has Neuroprotective Effects in a Rat Model of Cerebral Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) is an etiologic component of several neurologic pathologies associated to cognitive impairment. The mechanisms involved in HI-induced tissue damage start immediately after HI and extend for days. Acetylcholine is an important neurotransmitter in the central nervous system and exerts a protector effect on tissue damage by modulating inflammation, and cholinesterase inhibitors have shown neuroprotective properties and their action are often attributed to inhibition of the immune response. The administration of Huperzia quadrifariata alkaloid extract (HqAE), with potent and selective cholinesterase inhibitor properties, will reduce the HI induced behavioral deficits and tissue damage. A total of 84 newborn Wistar rat pups at post natal day 7 (PND7) were subjected to right carotid occlusion followed by 1 h of hypoxia (8% of O2) and i.p. injections of saline, vehicle or HqAE (10 mg/kg). Morris Water Maze and inhibitory avoidance tests were used to assess the cognitive function. Flow cytometry was performed at PND11. Histological analysis was performed at PND45. HqAE treatment was able to prevent the HI induced cognitive deficits in both tests and, at PND45, histological analysis showed that HqAE treatment reduced hippocampus tissue damage. Flow cytometry of the injured hippocampus revealed that the treatment was able to reduce cellular death and the number of infiltrating T cells. Altogether, these results show the therapeutic potential of the Huperzia quadrifariata alkaloid extract to prevent cognitive deficits and histological damage caused by neonatal hypoxia-ischemia, probably by reducing cellular death and T cell mobilization.

Effect of Sciatic Nerve Transection on acetylcholinesterase activity in spinal cord and skeletal muscles of the bullfrog Lithobates catesbeianus

Abstract Sciatic nerve transection (SNT), a model for studying neuropathic pain, mimics the clinical symptoms of phantom limb, a pain condition that arises in humans after amputation or transverse spinal lesions. In some vertebrate tissues, this condition decreases acetylcholinesterase (AChE) activity, the enzyme responsible for fast hydrolysis of released acetylcholine in cholinergic synapses. In spinal cord of frog Rana pipiens, this enzymes activity was not significantly changed in the first days following ventral root transection, another model for studying neuropathic pain. An answerable question is whether SNT decreases AChE activity in spinal cord of frog Lithobates catesbeianus, a species that has been used as a model for studying SNT-induced neuropathic pain. Since each animal model has been created with a specific methodology, and the findings tend to vary widely with slight changes in the method used to induce pain, our study assessed AChE activity 3 and 10 days after complete SNT in lumbosacral spinal cord of adult male bullfrog Lithobates catesbeianus. Because there are time scale differences of motor endplate maturation in rat skeletal muscles, our study also measured the AChE activity in bullfrog tibial posticus (a postural muscle) and gastrocnemius (a typical skeletal muscle that is frequently used to study the motor system) muscles. AChE activity did not show significant changes 3 and 10 days following SNT in spinal cord. Also, no significant change occurred in AChE activity in tibial posticus and gastrocnemius muscles at day 3. However, a significant decrease was found at day 10, with reductions of 18% and 20% in tibial posticus and gastrocnemius, respectively. At present we cannot explain this change in AChE activity. While temporally different, the direction of the change was similar to that described for rats. This similarity indicates that bullfrog is a valid model for investigating AChE activity following SNT.

Resumo A transecção do nervo isquiático (SNT), um modelo para estudar dor neuropática, simula os sintomas clínicos do membro fantasma, uma condição dolorosa que ocorre nos humanos após amputação ou secção completa da medula espinal. Essa condição muda a atividade da acetilcolinesterase (AChE), a enzima responsável pela rápida hidrólise da acetilcolina liberada nas sinapses colinérgicas, em alguns tecidos de vertebrados. Em medula espinal de rã Rana pipiens, a atividade da AChE não foi significativamente alterada nos primeiros dias após a secção da raiz ventral, outro modelo para o estudo da dor neuropática. Uma questão ainda não respondida é se a SNT diminui a atividade da AChE na medula espinal de rã Lithobates catesbeianus, uma espécie que vem sendo usada como modelo em estudos da dor neuropática induzida por SNT. Como cada modelo animal é criado a partir de metodologia específica, e seus resultados tendem a variar com pequenas mudanças na metodologia de indução da dor, o presente estudo avaliou a atividade da AChE em medula espinal lombossacral de rã-touro Lithobates catesbeianus, adultos, machos, 3 e 10 dias após a completa SNT. Como há diferenças temporais na maturação de placas motoras em músculos esqueléticos de ratos, nosso estudo ainda demonstrou, na rã-touro, os efeitos da SNT sobre a atividade da AChE nos músculos esqueléticos tibial posticus, um músculo postural, e gastrocnêmio, um músculo frequentemente usado em estudos do sistema motor. A atividade da AChE não mudou significativamente na medula espinal aos 3 e 10 dias após a SNT. Nos músculos, a atividade não alterou significativamente aos 3 dias após a lesão, mas reduziu de forma significativa aos 10 dias após a SNT. Aos 10 dias, a diminuição foi 18% no músculo tibial posticus e 20% no gastrocnêmio. No momento, nós não temos explicação para essa mudança na atividade da AChE. Embora temporalmente diferente, o sentido da mudança é similar ao que é descrito em ratos. Esta similaridade torna a rã-touro um modelo válido para se estudar questões ainda não respondidas da SNT sobre a AChE.

Irish GP attitudes towards sickness certification and the 'fit note'.

BACKGROUND: Sickness certification poses significant challenges to most general practitioners (GPs). As an alternative form of certification, in 2010, the UK implemented the 'fit note', which focuses on returning to work after illness. It has been well received in the UK but little is known of non-UK GP attitudes towards the fit note. AIMS: To evaluate the challenges Irish GPs experience with the current sickness certification system and their attitudes towards the fit note. METHODS: A questionnaire assessing attitudes, practices and preferences regarding the fit note was developed, piloted and posted to a representative sample of Irish GPs selected by systematic sampling. Descriptive statistics, chi-square tests and binary logistic regression were used to analyse the results. RESULTS: Of 305 GPs surveyed, 64% (196) completed the survey. Ninety per cent (177) of responders reported a lack of available rehabilitation services for patients on sick leave and 66% (124) reported adverse effects of sickness certification on therapeutic relationships with patients. Those who indicated a preference for introducing the fit note (53%; 104) were significantly more likely to endorse both an excessive focus on disability [odds ratio (OR) = 3.16] and lack of GP training (OR = 2.04) in the sickness certification process. CONCLUSIONS: Difficulties associated with sickness certification are apparent at GP-patient, GP-employer and GP-health service levels. Addressing these will require intervention at each of these levels and may be facilitated by challenging the assumption that illness equates to disability.

Environmental stimulation improves performance in the ox-maze task and recovers Na+,K+-ATPase activity in the hippocampus of hypoxic-ischemic rats.

In animal models, environmental enrichment (EE) has been found to be an efficient treatment for alleviating the consequences of neonatal hypoxia-ischemia (HI). However the potential for this therapeutic strategy and the mechanisms involved are not yet clear. The aim of present study is to investigate behavioral performance in the ox-maze test and Na+,K+-ATPase, catalase (CAT) and glutathione peroxidase (GPx) activities in the hippocampus of rats that suffered neonatal HI and were stimulated in an enriched environment. Seven-day-old rats were submitted to the HI procedure and divided into four groups: control maintained in standard environment (CTSE), control submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Animals were stimulated with EE for 9 weeks (1 h/day for 6 days/week) and then behavioral and biochemical parameters were evaluated. Present results indicate learning and memory in the ox-maze task were impaired in HI rats and this effect was recovered after EE. Hypoxic-ischemic event did not alter the Na+,K+-ATPase activity in the right hippocampus (ipsilateral to arterial occlusion). However, on the contralateral hemisphere, HI caused a decrease in this enzyme activity that was recovered by EE. The activities of GPx and CAT were not changed by HI in any group evaluated. In conclusion, EE was effective in recovering learning and memory impairment in the ox-maze task and Na+,K+-ATPase activity in the hippocampus caused by HI. The present data provide further support for the therapeutic potential of environmental stimulation after neonatal HI in rats.

Autophagy in the brain of neonates following hypoxia-ischemia shows sex- and region-specific effects.

Autophagy is responsible for the bulk degradation of cytoplasmic contents including organelles through the lysosomal machinery. Neonatal hypoxia-ischemia (HI) causes cell death in the brain by caspase-dependent and independent pathways. Ischemic insults also increase the formation of autophagosomes and activate autophagy. This study assessed the possible sex- and region-specific differences of autophagy activity in neonates subjected to HI brain injury. HI males had a modest decrease in lysosome numbers with no effect on LC3B-II protein in the cortex. In contrast, HI females had decreased lysosome numbers and their LC3B-II protein expression was significantly increased in the cortex following HI. In the hippocampus, both HI males and all females had increased numbers of autolysosomes suggesting activation of autophagy but with no effect on lysosome numbers, or Beclin-1 or LC3B protein levels. Males and females had increases in caspase 3/7 activity in their cortices and hippocampi following HI, though the increases were three to sixfold greater in females. The present data: (a) confirm greater caspase activation in the brains of females compared to males following HI; (b) suggest a partial failure to degrade LC3B-II protein in cortical but not hippocampal lysosomes of females as compared to males following neonatal HI; (c) all females have greater basal autophagy activity than males which may protect cells against injury by increasing cell turnover and (d) demonstrate that autophagy pathways are disturbed in regional- and sex-specific patterns in the rat brain following neonatal HI.

Proline-induced changes in acetylcholinesterase activity and gene expression in zebrafish brain: reversal by antipsychotic drugs.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

Evidences that maternal swimming exercise improves antioxidant defenses and induces mitochondrial biogenesis in the brain of young Wistar rats.

Physical exercise during pregnancy has been considered beneficial to mother and child. Recent studies showed that maternal swimming improves memory in the offspring, increases hippocampal neurogenesis and levels of neurotrophic factors. The objective of this work was to investigate the effect of maternal swimming during pregnancy on redox status and mitochondrial parameters in brain structures from the offspring. Adult female Wistar rats were submitted to five swimming sessions (30 min/day) prior to mating with adult male Wistar rats, and then trained during the pregnancy (five sessions of 30-min swimming/week). The litter was sacrificed when 7 days old, when cerebellum, parietal cortex, hippocampus, and striatum were dissected. We evaluated the production of reactive species and antioxidant status, measuring the activities of superoxide-dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx), as well as non-enzymatic antioxidants. We also investigated a potential mitochondrial biogenesis regarding mitochondrion mass and membrane potential, through cytometric approaches. Our results showed that maternal swimming exercise promoted an increase in reactive species levels in cerebellum, parietal cortex, and hippocampus, demonstrated by an increase in dichlorofluorescein oxidation. Mitochondrial superoxide was reduced in cerebellum and parietal cortex, while nitrite levels were increased in cerebellum, parietal cortex, hippocampus, and striatum. Antioxidant status was improved in cerebellum, parietal cortex, and hippocampus. SOD activity was increased in parietal cortex, and was not altered in the remaining brain structures. CAT and GPx activities, as well as non-enzymatic antioxidant potential, were increased in cerebellum, parietal cortex, and hippocampus of rats whose mothers were exercised. Finally, we observed an increased mitochondrial mass and membrane potential, suggesting mitochondriogenesis, in cerebellum and parietal cortex of pups subjected to maternal swimming. In conclusion, maternal swimming exercise induced neurometabolic programing in the offspring that could be of benefit to the rats against future cerebral insults.