Therapeutic hypothermia for the treatment of neonatal hypoxia-ischemia: sex-dependent modulation of reactive astrogliosis.

Therapeutic hypothermia (TH) is the standard treatment for neonatal hypoxia-ischemia (HI) with a time window limited up to 6 h post injury. However, influence of sexual dimorphism in the therapeutic window for TH has not yet been elucidated in animal models of HI. Therefore, the aim of this study was to investigate the most effective time window to start TH in male and female rats submitted to neonatal HI. Wistar rats (P7) were divided into the following groups: NAÏVE and SHAM (control groups), HI (submitted to HI) and TH (submitted to HI and TH; 32ºC for 5 h). TH was started at 2 h (TH-2 h group), 4 h (TH-4 h group), or 6 h (TH-6 h group) after HI. At P14, animals were subjected to behavioural tests, volume of lesion and reactive astrogliosis assessments. Male and female rats from the TH-2 h group showed reduction in the latency of behavioral tests, and decrease in volume of lesion and intensity of GFAP immunofluorescence. TH-2 h females also showed reduction of degenerative cells and morphological changes in astrocytes. Interestingly, females from the TH-6 h group showed an increase in volume of lesion and in number of degenerative hippocampal cells, associated with worse behavioral performance. Together, these results indicate that TH neuroprotection is time- and sex-dependent. Moreover, TH started later (6 h) can worsen volume of brain lesion in females. These data indicate the need to develop specific therapeutic protocols for each sex and reinforce the importance of early onset of the hypothermic treatment.

Neuroprotective Role of Lactoferrin during Early Brain Development and Injury through Lifespan.

Early adverse fetal environments can significantly disturb central nervous system (CNS) development and subsequently alter brain maturation. Nutritional status is a major variable to be considered during development and increasing evidence links neonate and preterm infant impaired brain growth with neurological and psychiatric diseases in adulthood. Breastfeeding is one of the main components required for healthy newborn development due to the many "constitutive" elements breastmilk contains. Maternal intake of specific nutrients during lactation may alter milk composition, thus affecting newborn nutrition and, potentially, brain development. Lactoferrin (Lf) is a major protein present in colostrum and the main protein in human milk, which plays an important role in the benefits of breastfeeding during postnatal development. It has been demonstrated that Lf has antimicrobial, as well as anti-inflammatory properties, and is potentially able to reduce the incidence of sepsis and necrotizing enterocolitis (NEC), which are particularly frequent in premature births. The anti-inflammatory effects of Lf can reduce birth-related pathologies by decreasing the release of pro-inflammatory factors and inhibiting premature cervix maturation (also related to commensal microbiome abnormalities) that could contribute to disrupting brain development. Pre-clinical evidence shows that Lf protects the developing brain from neuronal injury, enhances brain connectivity and neurotrophin production, and decreases inflammation in models of perinatal inflammatory challenge, intrauterine growth restriction (IUGR) and neonatal hypoxia-ischemia (HI). In this context, Lf can provide nutritional support for brain development and cognition and prevent the origin of neuropsychiatric diseases later in life. In this narrative review, we consider the role of certain nutrients during neurodevelopment linking to the latest research on lactoferrin with respect to neonatology. We also discuss new evidence indicating that early neuroprotective pathways modulated by Lf could prevent neurodegeneration through anti-inflammatory and immunomodulatory processes.

Early environmental enrichment rescues memory impairments provoked by mild neonatal hypoxia-ischemia in adolescent mice.

Hypoxia-ischemia (HI) is a consequence of a lack of oxygen and glucose support to the developing brain, which causes several neurodevelopmental impairments. Environmental enrichment (EE) is considered an option to recover the alterations observed in rodents exposed to HI. The aim of this study was to investigate the impact of early EE on memory, hippocampal volume and brain-derived neurotrophic factor (Bbnf) and glucocorticoid receptor (Nr3c1) gene expression of mice exposed to HI. At P10, pups underwent right carotid artery permanent occlusion followed by 35 min of 8% O2 hypoxic environment. Starting at P11, animals were reared in EE or in standard cage (HI-SC or SHAM-SC) conditions until behavioral testing (P45). SHAM pups did not undergo carotid ligation and hypoxic exposure. Memory performance was assessed in the Y-maze, Novel object recognition, and Barnes maze. Animals were then sacrificed for analysis of hippocampal volume and Bdnf and Nr3c1 gene expression. We observed that animals exposed to HI performed worse in all three tests compared to SHAM animals. Furthermore, HI animals exposed to EE did not differ from SHAM animals in all tasks. Moreover, HI decreased hippocampal volume, while animals reared in early EE were not different compared to SHAM animals. Animals exposed to HI also showed upregulated hippocampal Bdnf expression compared to SHAM animals. We conclude that early EE from P11 to P45 proved to be effective in recovering memory impairments and hippocampal volume loss elicited by HI. Nevertheless, Bdnf expression was not associated with the improvements in memory performance observed in animals exposed to EE after a hypoxic-ischemic event.

Pre- and early postnatal enriched environmental experiences prevent neonatal hypoxia-ischemia late neurodegeneration via metabolic and neuroplastic mechanisms.

Prenatal and early postnatal periods are important for brain development and neural function. Neonatal insults such as hypoxia-ischemia (HI) causes prolonged neural and metabolic dysregulation, affecting central nervous system maturation. There is evidence that brain hypometabolism could increase the risk of adult-onset neurodegenerative diseases. However, the impact of non-pharmacologic strategies to attenuate HI-induced brain glucose dysfunction is still underexplored. This study investigated the long-term effects of early environmental enrichment in metabolic, cell, and functional responses after neonatal HI. Thereby, male Wistar rats were divided according to surgical procedure, sham, and HI (performed at postnatal day 3), and the allocation to standard (SC) or enriched condition (EC) during gestation and lactation periods. In-vivo cerebral metabolism was assessed by means of [18 F]-FDG micro-positron emission tomography, and cognitive, biochemical, and histological analyses were performed in adulthood. Our findings reveal that HI causes a reduction in glucose metabolism and glucose transporter levels as well as hyposynchronicity in metabolic brain networks. However, EC during prenatal or early postnatal period attenuated these metabolic disturbances. A positive correlation was observed between [18 F]-FDG values and volume ratios in adulthood, indicating that preserved tissue by EC is metabolically active. EC promotes better cognitive scores, as well as down-regulation of amyloid precursor protein in the parietal cortex and hippocampus of HI animals. Furthermore, growth-associated protein 43 was up-regulated in the cortex of EC animals. Altogether, results presented support that EC during gestation and lactation period can reduce HI-induced impairments that may contribute to functional decline and progressive late neurodegeneration.

Phytoestrogen coumestrol attenuates brain mitochondrial dysfunction and long-term cognitive deficits following neonatal hypoxia-ischemia.

INTRODUCTION: Neonatal Hypoxia-Ischemia (HI) is a major cause of morbidity and mortality, and is frequently associated with short and long-term neurologic and cognitive impairments. The HI injury causes mitochondrial damage leading to increased production of reactive oxygen species (ROS). Phytoestrogens are non-steroidal plant substances structurally and functionally similar to estrogen. Coumestrol is a potent isoflavonoid with a protective effect against ischemic brain damage in adult rats. Our aim was to determine if coumestrol treatment following neonatal HI attenuates the long-term cognitive deficits induced by neonatal HI, as well as to investigate one possible mechanism underlying its potential effect. METHODS: On the 7th postnatal day, male Wistar rats were submitted to the Levine-Rice HI model. Intraperitoneal injections of 20 mg/kg of coumestrol, or vehicle, were administered immediately pre-hypoxia or 3 h post-hypoxia. At 12 h after HI the mitochondrial status and ROS levels were determined. At 60th postnatal day the cognitive deficits were revealed in the Morris water maze reference and working spatial memories. Following behavioral analysis, histological assessment was performed and reactive astrogliosis was measured by GFAP expression. RESULTS: Results demonstrate that both pre- and post-HI administration of coumestrol were able to counteract the long-term cognitive and morphological impairments caused by HI, as well as to block the late reactive astrogliosis. The pre-HI administration of coumestrol was able to prevent the early mitochondrial dysfunction in the hippocampus of injured rat pups. CONCLUSION: Present data suggest that coumestrol exerts protection against experimental neonatal brain hypoxia-ischemia through, at least in part, early modulation of mitochondrial function.

Stem Cells from Human Exfoliated Deciduous Teeth Modulate Early Astrocyte Response after Spinal Cord Contusion.

The transplantation of stem cells from human exfoliated deciduous teeth (SHED) has been studied as a possible treatment strategy for spinal cord injuries (SCIs) due to its potential for promoting tissue protection and functional recovery. The aim of the present study was to investigate the effects of the early transplantation of SHED on glial scar formation and astrocytic reaction after an experimental model of SCI. Wistar rats were spinalized using the NYU Impactor. Animals were randomly distributed into three groups: control (naive) (animal with no manipulation); SCI (receiving laminectomy followed by SCI and treated with vehicle), and SHED (SCI rat treated with intraspinal SHED transplantation, 1 h after SCI). In vitro investigation demonstrated that SHED were able to express mesenchymal stem cells, vimentin and S100B markers, related with neural progenitor and glial cells, respectively. The acute SHED transplantation promoted functional recovery, measured as from the first week after spinal cord contusion by Basso, Beattie, and Bresnahan scale. Twenty-four and 48 h after lesion, flow cytometry revealed a spinal cord vimentin+ cells increment in the SHED group. The increase of vimentin+ cells was confirmed by immunofluorescence. Moreover, the bioavailability of astrocytic proteins such as S100B and Kir4.1 shown to be increased in the spinal cord of SHED group, whereas there was a glial scar reduction, as indicated by ELISA and Western blot techniques. The presented results support that SHED act as a neuroprotector agent after transplantation, probably through paracrine signaling to reduce glial scar formation, inducing tissue plasticity and functional recovery.

Correction to: Stem Cells from Human Exfoliated Deciduous Teeth Modulate Early Astrocyte Response after Spinal Cord Contusion.

The authors hereby declare that the Figure 4 in page eight of the paper "Stem cells from human exfoliated deciduous teeth modulate early astrocyte response after spinal cord contusion" authored by Fabrício Nicola and colleagues (DOI: 10.1007/s12035-018-1127-4) was mistakenly included.

Poly (lactide-co-glycolide) (PLGA) Scaffold Induces Short-term Nerve Regeneration and Functional Recovery Following Sciatic Nerve Transection in Rats.

Peripheral nerve injury is an important cause of incapability and has limited available treatment. Autologous donor nerve implant is the golden standard treatment, however, may cause secondary deficits. Stem cells show positive results in preclinical settings, preserving tissue and function. We tested the efficacy of stem cells derived from human exfoliated deciduous teeth seeded in poly (lactide-co-glycolide) scaffolds in sciatic nerve transection model. Seventy-two adult male Wistar rats had 7-mm nerve gap bridge using scaffolds with (or without) stem cells. Animals were randomly divided into: sham-operated; sham-operated without scaffold; sham-operated + scaffold + stem cells; sciatic transection + no treatment; sciatic transection + acellular scaffolds; sciatic transection + scaffold + stem cells. Sciatic Functional Index and Ladder Rung Walking tests were performed before (-1), 14 and 28 days after surgery. Morphometric nerve measurement and muscle weights were assessed. Scaffolds with stem cells improved function in Sciatic Functional Index. Acellular scaffold was effective, promoting functional recovery and nerve regeneration following nerve injury. Scaffolds provide better nerve regeneration and functional recovery after sciatic transection. Despite cell therapy promoting faster recovery after sciatic transection in the Sciatic Index Score, stem cells did not improve functional and morphological recovery after nerve injury. This is the first study testing the potential use of scaffolds combined with stem cells in the early stages after injury. Scaffolds with stem cells could accelerate nerve recovery and favor adjuvant therapies, evidencing the need for further studies to increase the knowledge about stem cells' mechanisms.

Intracardiac Injection of Dental Pulp Stem Cells After Neonatal Hypoxia-Ischemia Prevents Cognitive Deficits in Rats.

Neonatal hypoxia-ischemia (HI) is associated to cognitive and motor impairments and until the moment there is no proven treatment. The underlying neuroprotective mechanisms of stem cells are partially understood and include decrease in excitotoxicity, apoptosis and inflammation suppression. This study was conducted in order to test the effects of intracardiac transplantation of human dental pulp stem cells (hDPSCs) for treating HI damage. Seven-day-old Wistar rats were divided into four groups: sham-saline, sham-hDPSCs, HI-saline, and HI-hDPSCs. Motor and cognitive tasks were performed from postnatal day 30. HI-induced cognitive deficits in the novel-object recognition test and in spatial reference memory impairment which were prevented by hDPSCs. No motor impairments were observed in HI animals. Immunofluorescence analysis showed human-positive nuclei in hDPSC-treated animals closely associated with anti-GFAP staining in the lesion scar tissue, suggesting that these cells were able to migrate to the injury site and could be providing support to CNS cells. Our study evidence novel evidence that hDPSC can contribute to the recovery following hypoxia-ischemia and highlight the need of further investigation in order to better understand the exact mechanisms underlying its neuroprotective effects.

Effects of progesterone on the neonatal brain following hypoxia-ischemia.

Progesterone displays a strong potential for the treatment of neonatal hypoxic-ischemic encephalopathy since it has been shown to be beneficial in the treatment of the central nervous system injuries in adult animals. Here, we evaluated the effects of the administration of progesterone (10 mg/kg) in seven-days-old male Wistar rats submitted to neonatal hypoxia-ischemia (HI). Progesterone was administered immediately before ischemia and/or 6 and 24 h after the onset of hypoxia. The body weight of the animals, the volume of brain lesion and the expression of p-Akt and procaspase-3 in the hippocampus were evaluated. All animals submitted to HI showed a reduction in the body weight. However, this reduction was more remarkable in those animals which received progesterone before surgery. Administration of progesterone was unable to reduce the volume of brain damage caused by HI. Moreover, no significant differences were observed in the expression of p-Akt and procaspase-3 in animals submitted to HI and treated with either progesterone or vehicle. In summary, progesterone did not show a neuroprotective effect on the volume of brain lesion in neonatal rats submitted to hypoxia-ischemia. Furthermore, progesterone was unable to modulate p-Akt and procaspase-3 signaling pathways, which may explain the absence of neuroprotection. On the other hand, it seems that administration of progesterone before ischemia exerts some systemic effect, leading to a remarkable reduction in the body weight.

Pregnancy swimming causes short- and long-term neuroprotection against hypoxia-ischemia in very immature rats.

BackgroundHypoxia-ischemia (HI) is a major cause of neurological damage in preterm newborn. Swimming during pregnancy alters the offspring's brain development. We tested the effects of swimming during pregnancy in the very immature rat brain.MethodsFemale Wistar rats (n=12) were assigned to the sedentary (SE, n=6) or the swimming (SW, n=6) group. From gestational day 0 (GD0) to GD21 the rats in the SW group were made to swim for 20 min/day. HI on postnatal day (PND) 3 rats caused sensorimotor and cognitive impairments. Animals were distributed into SE sham (SESH), sedentary HIP3 (SEHI), swimming sham (SWSH), and swimming HIP3 (SWHI) groups. At PND4 and PND5, Na+/K+-ATPase activity and brain-derived neurotrophic factor (BDNF) levels were assessed. During lactation and adulthood, neurological reflexes, sensorimotor, anxiety-related, and cognitive evaluations were made, followed by histological assessment at PND60.ResultsAt early stages, swimming caused an increase in hippocampal BDNF levels and in the maintenance of Na+/K+-ATPase function in the SWHI group. The SWHI group showed smaller lesions and the preservation of white matter tracts. SEHI animals showed a delay in reflex maturation, which was reverted in the SWHI group. HIP3 induced spatial memory deficits and hypomyelination in SEHI rats, which was reverted in the SWHI group.ConclusionSwimming during pregnancy neuroprotected the brains against HI in very immature neonatal rats.

Forced Treadmill Exercise Prevents Spatial Memory Deficits in Aged Rats Probably Through the Activation of Na+, K+-ATPase in the Hippocampus.

Regular physical activity has shown to improve the quality of life and to prevent age-related memory deficits. Memory processing requires proper regulation of several enzymes such as sodium-potassium adenosine triphosphatase (Na+, K+-ATPase) and acetylcholinesterase (AChE), which have a pivotal role in neuronal transmission. The present study investigated the effects of a treadmill running protocol in young (3 months), mature (6 months) and aged (22 months) Wistar rats, on: (a) cognitive function, as assessed in the Water maze spatial tasks; (b) Na+, K+-ATPase and AChE activities in the hippocampus following cognitive training alone or treadmill running combined with cognitive training. Animals of all ages were assigned to naïve (with no behavioral or exercise training), sedentary (non-exercised, with cognitive training) and exercised (20 min of daily running sessions, 3 times per week for 4 weeks and with cognitive training) groups. Cognition was assessed by reference and working memory tasks run in the Morris Water maze; 24 h after last session of behavioral testing, hippocampi were collected for biochemical analysis. Results demonstrated that: (a) a moderate treadmill running exercise prevented spatial learning and memory deficits in aged rats; (b) training in the Water maze increased both Na+, K+-ATPase and AChE activities in the hippocampus of mature and aged rats; (c) aged exercised rats displayed an even further increase of Na+, K+-ATPase activity in the hippocampus, (d) enzyme activity correlated with memory performance in aged rats. It is suggested that exercise prevents spatial memory deficits in aged rats probably through the activation of Na+, K+-ATPase in the hippocampus.

D-Galactose Causes Motor Coordination Impairment, and Histological and Biochemical Changes in the Cerebellum of Rats.

Classical galactosemia is an inborn error of carbohydrate metabolism in which patients accumulate high concentration of galactose in the brain. The most common treatment is a galactose-restricted diet. However, even treated patients develop several complications. One of the most common symptoms is motor coordination impairment, including affected gait, balance, and speech, as well as tremor and ataxia. In the present study, we investigated the effects of intracerebroventricular galactose administration on motor coordination, as well as on histological and biochemical parameters in cerebellum of adult rats. Wistar rats received 5 µL of galactose (4 mM) or saline by intracerebroventricular injection. The animals performed the beam walking test at 1 and 24 h after galactose administration. Histological and biochemical parameters were performed 24 h after the injections. The results showed motor coordination impairment at 24 h after galactose injection. Galactose also decreased the number of cells in the molecular and granular layers of the cerebellum. The immunohistochemistry results suggest that the cell types lost by galactose are neurons and astrocytes in the spinocerebellum and neurons in the cerebrocerebellum. Galactose increased active caspase-3 immunocontent and acetylcholinesterase activity, decreased acetylcholinesterase immunocontent, glutathione, and BDNF levels, as well as caused protein and DNA damage. Our results suggest that galactose induces histological and biochemical changes in cerebellum, which can be associated with motor coordination impairment.

Polymethylmethacrylate imbedded with antibiotics cranioplasty: An infection solution for moderate and large defects reconstruction?

BACKGROUND: In cases where autologous bone graft reconstruction is not possible (such as comminuted fractures, bone graft reabsorption, or infection) and the use of synthetic material is required, polymethylmethacrylate (PMMA) use is a safe and efficient solution. Studies comparing the incidence of postoperative complications between autologous and synthetic cranioplasty are heterogeneous, not allowing a conclusion of which is the best material for skull defects reconstruction. Current medical literature lacks prospective well-delineated studies with long-term follow-up that analyze the impact of antibiotic use in PMMA cranial reconstruction of moderate and large defects. METHODS: A prospective series of patients, who underwent cranioplasty reconstruction with PMMA impregnated with antibiotic, were followed for 2 years. Authors collected data regarding demographic status, clinical conditions, surgical information, and its complications. RESULTS: A total of 58 patients completed full follow-up with a mean group age of 40 years and a male predominance (77%). Major complications that required surgical management were identified in 5 patients, and 10 patients evolved with minor complications. Postoperative surgical site infection incidence was 3.2%. CONCLUSION: The infection rate in patients submitted to PMMA flap cranioplasty impregnated with antibiotic is significantly inferior comparing to the data described in medical literature. A lower infection incidence impacts secondary endpoints such as minimizing surgical morbidity, mortality, hospitalization period, and, consequently, costs.

Intracerebroventricular D-galactose administration impairs memory and alters activity and expression of acetylcholinesterase in the rat.

Tissue accumulation of galactose is a hallmark in classical galactosemia. Cognitive deficit is a symptom of this disease which is poorly understood. The aim of this study was to investigate the effects of intracerebroventricular administration of galactose on memory (inhibitory avoidance and novel object recognition tasks) of adult rats. We also investigated the effects of galactose on acetylcholinesterase (AChE) activity, immunocontent and gene expression in hippocampus and cerebral cortex. Wistar rats received a single injection of galactose (4mM) or saline (control). For behavioral parameters, galactose was injected 1h or 24h previously to the testing. For biochemical assessment, animals were decapitated 1h, 3h or 24h after galactose or saline injection; hippocampus and cerebral cortex were dissected. Results showed that galactose impairs the memory formation process in aversive memory (inhibitory avoidance task) and recognition memory (novel object recognition task) in rats. The activity of AChE was increased, whereas the gene expression of this enzyme was decreased in hippocampus, but not in cerebral cortex. These findings suggest that these changes in AChE may, at least in part, to lead to memory impairment caused by galactose. Taken together, our results can help understand the etiopathology of classical galactosemia.

Exposition to tannery wastewater did not alter behavioral and biochemical parameters in Wistar rats.

There are scarce data on the neurotoxicity in mammalian induced by tannery wastewaters. Previously, the anxiogenic effect of tannery wastewater was demonstrated in mice, while wastewater submitted to photoelectrooxidation (PEO) process treatment did not affect the anxiety state. Considering that species may response differently to xenobiotics, the aim of the present work was to study the effects of exposure to tannery wastewaters (non-PEO or PEO-treated) on behavioral and neurochemical markers in another species of laboratory animals, specifically Wistar rats. Male Wistar rats were given free access to water bottles containing non-PEO or PEO-treated tannery wastewaters (0.1, 1 and 5% in drinking water). During the exposure, behavioral tests of anxiety (elevated plus-maze, neophobia, open field and light-dark box), depression (forced swimming) and memory (inhibitory avoidance, novel object and discriminative avoidance) were performed. On the 30th day, brain structures were dissected out to evaluate cellular oxidative state (hippocampus, cerebellum and striatum) and acetylcholinesterase activity (hippocampus and striatum). Exposure to tannery effluent with or without photoelectrochemical treatment did not alter any behavioral and neurochemical parameters evaluated. Our data indicate that Wistar rats may not be an adequate species for ecotoxicological studies involving tannery effluents and that POE treatment did not generate other toxic compounds.

Effects of pre- and postnatal protein malnutrition in hypoxic-ischemic rats.

Neonatal hypoxic-ischemic encephalopathy (HI) is a major cause of nervous system damage and neurological morbidity. Perinatal malnutrition affects morphological, biochemical and behavioral aspects of neural development, including pathophysiological cascades of cell death triggered by ischemic events, so modifying resulting brain damage. Female Wistar rats were subjected to protein restriction during pregnancy and lactation (control group: 25% soybean protein; malnourished group: 7%). Seven days after delivery (PND7), their offspring were submitted to unilateral cerebral HI; rats were then tested for sensorimotor (PND7 and PND60) and memory (PND60) functions. Offspring of malnourished mothers showed marked reduction in body weight starting in lactation and persisting during the entire period of observation. There was a greater sensorimotor deficit after HI in malnourished (M) animals, in righting reflex and in home bedding task, indicating an interaction between diet and hypoxia-ischemia. At PND60, HI rats showed impaired performance when compared to controls in training and test sessions of rota-rod task, however there was no effect of malnutrition per se. In the open field, nourished HI (HI-N) presented an increase in crossings number; this effect was not present in HI-M group. Surprisingly, HI-M rats presented a better performance in inhibitory avoidance task and a smaller hemispheric brain damage as compared to HI-N animals. Our data points to a possible metabolic adaptation in hypoxic-ischemic animals receiving protein malnutrition during pregnancy and lactation; apparently we observed a neuroprotective effect of diet, possibly decreasing the brain energy demand, under a hypoxic-ischemic situation.