Increased number of excitatory synapsis and decreased number of inhibitory synapsis in the prefrontal cortex in autism.

Previous studies in autism spectrum disorder demonstrated an increased number of excitatory pyramidal cells and a decreased number of inhibitory parvalbumin+ chandelier interneurons in the prefrontal cortex of postmortem brains. How these changes in cellular composition affect the overall abundance of excitatory and inhibitory synapses in the cortex is not known. Herein, we quantified the number of excitatory and inhibitory synapses in the prefrontal cortex of 10 postmortem autism spectrum disorder brains and 10 control cases. To identify excitatory synapses, we used VGlut1 as a marker of the presynaptic component and postsynaptic density protein-95 as marker of the postsynaptic component. To identify inhibitory synapses, we used the vesicular gamma-aminobutyric acid transporter as a marker of the presynaptic component and gephyrin as a marker of the postsynaptic component. We used Puncta Analyzer to quantify the number of co-localized pre- and postsynaptic synaptic components in each area of interest. We found an increase in the number of excitatory synapses in upper cortical layers and a decrease in inhibitory synapses in all cortical layers in autism spectrum disorder brains compared with control cases. The alteration in the number of excitatory and inhibitory synapses could lead to neuronal dysfunction and disturbed network connectivity in the prefrontal cortex in autism spectrum disorder.

Pathology and Astrocytes in Autism.

A distinct pathology for autism spectrum disorder (ASD) remains elusive. Human and animal studies have focused on investigating the role of neurons in ASD. However, recent studies have hinted that glial cell pathology could be a characteristic of ASD. Astrocytes are the most abundant glial cell in the brain and play an important role in neuronal function, both during development and in adult. They regulate neuronal migration, dendritic and spine development, and control the concentration of neurotransmitters at the synaptic cleft. They are also responsible for synaptogenesis, synaptic development, and synaptic function. Therefore, any change in astrocyte number and/or function could contribute to the impairment of connectivity that has been reported in ASD. Data available to date is scarce but indicates that while the number of astrocytes is reduced, their state of activation and their GFAP expression is increased in ASD. Disruption of astrocyte function in ASD may affect proper neurotransmitter metabolism, synaptogenesis, and the state of brain inflammation. Astrocytes alterations are common to ASD and other neurodevelopmental disorders. Future studies about the role of astrocytes in ASD are required to better understand this disorder.

Decreased number and increased activation state of astrocytes in gray and white matter of the prefrontal cortex in autism.

The cerebral cortex presents with alterations in the number of specific cell types in autism spectrum disorder (ASD). Astrocytes have many functions in the brain including a role in higher cognitive functions and in inflammatory brain processes. Therefore, an alteration in number, function, and/or activation state of astrocytes, could be present in ASD. We quantified astrocyte number in the gray and white matter of the prefrontal cortex-BA9, BA46, and BA47-in 15 ASD and 15 age- and sex-matched control cases. We labeled astrocytes with antibodies against the protein GFAP and S100ß, markers of astrocytes. We found a significant decrease in the number of astrocytes in the gray and white matter of all prefrontal areas of interest with both markers. We also found an increased state of activation of GFAP+ astrocytes in all areas. A reduced number of astrocytes in the cerebral cortex in ASD could lead to impaired synaptic function and disrupted connectivity. An increased astrocyte activation may indicate a chronic mild inflammatory state of the cerebral cortex in ASD. Overall, we found that astrocytes are disrupted in ASD.

Chandelier Cartridge Density Is Reduced in the Prefrontal Cortex in Autism.

An alteration in the balance of excitation-inhibition has been proposed as a common characteristic of the cerebral cortex in autism, which may be due to an alteration in the number and/or function of the excitatory and/or inhibitory cells that form the cortical circuitry. We previously found a decreased number of the parvalbumin (PV)+ interneuron known as Chandelier (Ch) cell in the prefrontal cortex in autism. This decrease could result from a decreased number of Ch cells, but also from decreased PV protein expression by Ch cells. To further determine if Ch cell number is altered in autism, we quantified the number of Ch cells following a different approach and different patient cohort than in our previous studies. We quantified the number of Ch cell cartridges-rather than Ch cell somata-that expressed GAT1-rather than PV. Specifically, we quantified GAT1+ cartridges in prefrontal areas BA9, BA46, and BA47 of 11 cases with autism and 11 control cases. We found that the density of GAT1+ cartridges was decreased in autism in all areas and layers. Whether this alteration is cause or effect remains unclear but could result from alterations that take place during cortical prenatal and/or postnatal development.

Neuronal injury and death following focal mild brain injury: The role of network excitability and seizure.

OBJECTIVES: While traumatic brain injury (TBI) is a predisposing factor for development of post-traumatic epilepsy (PTE), the occurrence of seizures following brain trauma can infuriate adverse consequences of brain injury. However, the effect of seizures in epileptogenesis after mild TBI cannot yet be accurately confirmed. This study was designed to investigate the histopathological and molecular modifications induced by seizures on traumatized brain. MATERIALS AND METHODS: Using a new method, head was traumatized and seizures were evoked by sub-convulsive dose of pentylenetetrazole (PTZ) fifteen days after induction of focal mild TBI. Convulsion assessments were performed one hour after PTZ injection and was followed by histopathological and molecular evaluations. RESULTS: A significantly higher score and longer duration of seizure attacks as well as higher number of epileptiform discharges were observed in the TBI+PTZ group compared to sham and TBI groups. An elevated number of apoptotic cells was observed in the TBI+PTZ group compared to sham and TBI rats. Molecular investigations revealed higher levels of Bax/Bcl2 ratio, Caspase 3, and NF-κB in the TBI+PTZ group compared to the other animal groups. The value of Nrf2 did not change after mild TBI compared to sham and PTZ control groups. Occurrence of seizures after TBI, however, significantly decreased the level of Nrf2. CONCLUSION: Our data indicated that seizure occurrence following mild TBI aggravates cell injury and death via activation of neuroinflammatory processes and may increase the risk of PTE. Additionally, our results suggest a potential protective role of Nrf2 after chemically evoked PTE.

The fundamental building blocks of cortical development are established in human exencephaly.

BACKGROUND: The presence and status of progenitor/stem cells in excencephalic brain have not been previously examined. METHODS: Brain sections of excencephalic 17-week fetus were stained for specific stem and mature cell markers. RESULTS: The ventricles were open, the developing cerebral cortex was thin in the radial dimension, and the ventricular surface was undulated. There was a decreased ratio of subventricular/ventricular zone radial glia precursor cells (RGCs; PAX6+ and HOPX+ cells), a decreased number of intermediate progenitor cells (IPCs; TBR2+), a decreased number of neurons (MAP2+), and an increased number of astrocytes (S100b+), compared to the control. MAP2+ neurons, S100b+ astrocytes, and OLIG2+ oligodendrocytes were present within the subventricular zone. CONCLUSIONS: This indicates that the underlying condition did not initially preclude radial glial cells from undergoing asymmetric divisions that produce IPCs but halted the developmental progression. RGC and IPC presence in the developing cerebral cortex demonstrates that the fundamental building blocks of cortical formation had been established and that a normal sequence of developmental steps had been initiated in this case of exencephaly. These data expand our understanding of exencephaly etiology and highlight the status of cortical progenitor cells that may be linked to the disorder.

Progesterone modulates post-traumatic epileptogenesis through regulation of BDNF-TrkB signaling and cell survival-related pathways in the rat hippocampus.

Female sex hormone, progesterone, in addition to seizure modifying activity is also known as a potential protective agent against various brain injury conditions. Considering the predisposal role of traumatic brain injury (TBI) on developing post-traumatic epilepsy (PTE), the effect of progesterone on post-traumatic epileptogenesis is not investigated yet. Male Wistar rats were given a moderate focal weight drop injury (500 gr) or sham surgery and then progesterone (16 and 32mg/kg) was given daily for two consecutive weeks. On day 15 of injury, seizures were induced by administration of a GABAA receptor antagonist, pentylenetetrazole (PTZ, 30 mg/kg). Seizures were then assessed over a 1-h period using the Racine clinical rating scale. Traumatized animals that received 32 mg/kg progesterone had reduced score, duration of seizures and almost did not show tonic-clonic seizures during 60 min versus the untreated trauma group. In line with behavioral alterations, 32 mg/kg progesterone enhanced the amount of Nrf2 and HO-1 proteins and decreased the level of NF-kB, BDNF, Caspase 3 and ratio of Bax/Bcl-2 in the ipsilateral hippocampus. Additionally, the number of TUNEL-positive apoptotic cells, as well as injured dark neurons in the parietal cortex and hippocampal CA1 of 32 mg/kg-treated animals showed a significant reduction. Administration of 16 mg/kg progesterone elevated production of BDNF, Bax and Caspase 3 and decreased anti-apoptotic Bcl-2 protein. Taken together, an early administration of 32 mg/kg of progesterone after TBI for two weeks post-injury modified seizure activity. Our findings suggest that post-traumatic anti-epileptogenesis property of a high dose of progesterone partly occurs through the manipulation of BDNF-TrkB axis along with control of cell survival pathways.

Cortical interlaminar astrocytes across the therian mammal radiation.

Interlaminar astrocytes (ILA) in the cerebral cortex possess a soma in layer I and extend an interlaminar process that runs perpendicular to the pia into deeper cortical layers. We examined cerebral cortex from 46 species that encompassed most orders of therian mammalians, including 22 primate species. We described two distinct cell types with interlaminar processes that have been referred to as ILA, that we termed pial ILA and supial ILA. ILA subtypes differ in somatic morphology, position in layer I, and presence across species. We further described rudimentary ILA that have short GFAP+ processes that do not exit layer I, and "typical" ILA with longer GFAP+ processes that exit layer I. Pial ILA were present in all mammalian species analyzed, with typical ILA observed in Primates, Scandentia, Chiroptera, Carnivora, Artiodactyla, Hyracoidea, and Proboscidea. Subpial ILA were absent in Marsupialia, and typical subpial ILA were only found in Primate. We focused on the properties of pial ILA by investigating the molecular properties of pial ILA and confirming their astrocytic nature. We found that while the density of pial ILA somata only varied slightly, the complexity of ILA processes varied greatly across species. Primates, specifically bonobo, chimpanzee, orangutan, and human, exhibited pial ILA with the highest complexity. We showed that interlaminar processes contact neurons, pia, and capillaries, suggesting a potential role for ILA in the blood-brain barrier and facilitating communication among cortical neurons, astrocytes, capillaries, meninges, and cerebrospinal fluid.

Effect of Multiple Intraperitoneal Injections of Human Bone Marrow Mesenchymal Stem Cells on Cuprizone Model of Multiple Sclerosis

Background: Bone marrow mesenchymal stem cells (BM-MSCs) elicit neuroprotective effects, and their repair ability has been investigated in different experimental models. We aimed to investigate the effect of multiple i.p. BM-MSCs injections in the cuprizone model of multiple sclerosis in mice. Methods: Adult male C57BL/6 mice (n = 40) were fed a regular diet or a diet containing cuprizone (0.2% w/w) for 6 six weeks. Bone marrow samples were taken from patients with spinal cord injury. BM-MSCs (2 × 106 in 1 milliliter medium) were administered intraperitoneally for two consecutive weeks at the end of the forth weeks of cuprizone administration. Animals (n = 12) were perfused with 10% paraformaldehyde at the end of sixth week. The brains were sectioned coronally in 6-8-µm thickness (-2.3 to 1.8 mm from bregma). The sections were stained by luxol fast blue-cresyl violet, and images were captured via a microscope. Demyelination ratio was estimated in corpus callosum in a blind manner. A quantitative real-time PCR was used to measure the myelin basic protein gene expression at sixth week. Results: Histologically, cuprizone induced demyelination in the corpus callosum. Demyelinated area was diminished in the corpus callosum of cell-administered group. Cuprizone could decrease myelin-binding protein mRNAs expression in corpus callosum, which was significantly recovered after BM-MSCs injections. Conclusion: Our data indicated a remyelination potency of multiple i.p. BM-MSCs in the cuprizone model of multiple sclerosis in mice.

Low, but not high, dose triptolide controls neuroinflammation and improves behavioral deficits in toxic model of multiple sclerosis by dampening of NF-κB activation and acceleration of intrinsic myelin repair.

Cuprizone (Cup) is a copper chelating agent frequently used to study factors that affect oligodendrocytes (OLGs) death and acute demyelination. Triptolide (TP), a nuclear factor-kappaB (NF-κB) blocker, is a major bioactive component of Tripterygium wilfordii Hook f. (TWHf) with various therapeutic activities. In this study, we examined the effects of TP on neuroglia activation, inflammation, apoptosis, demyelination, and behavioral deficits in the Cup-induced toxic model of multiple sclerosis (MS). C57BL/6 J mice were fed with chow containing 0.2% Cup for 6 weeks to induce detectable neuroinflammation and myelin loss. TP was administered intraperitoneally at different doses (125, 250 or 500 µg/kg/day) during the last week of the Cup challenge. Although TP substantially decreased Cup-induced NF-κB extra activation, TNF-α and IL-1 over expression, and gliosis in a dose-dependent manner, only low dose of TP (TP-125) was able to raise the number of OLGs precursor cells (NG-2+/O4+), reduce Bax/Bcl-2 ratio and improve behavioral deficits. In addition, TP-125 decreased NF-κB activation on GFAP+ astrocytes more than MAC-3+ microglial and MOG+ oligodendrocytes which suggested the possibility of specific dampening of NF-κB signaling in reactive astrocytes. Behavioral assessments by open-field and rota-rod tests showed that only TP-125 notably improved motor function and motor coordination compared to the Cup group. These findings highlight the pivotal role of NF-κB signaling in the oligodendrogenesis and lesion reduction in demyelination diseases such as MS.

Enhancement of therapeutic DNA vaccine potency by melatonin through inhibiting VEGF expression and induction of antitumor immunity mediated by CD8+ T cells.

To be effective, therapeutic cancer vaccines should stimulate both an effective cell-mediated and a robust cytotoxic CD8+ T-cell response against human papillomavirus (HPV)-infected cells to treat the pre-existing tumors and prevent potential future tumors. In this study, the therapeutic experiments were designed in order to evaluate antitumor effect against the syngeneic TC-1 tumor model. The anti-tumor efficacy of a HPV-16 E7 DNA vaccine adjuvanted with melatonin (MLT) was evaluated in a C57BL/6 mouse tumor model by measuring tumor growth post vaccination and the survival rate of tumor-bearing mice, analyzing the specific lymphocyte proliferation responses in control and vaccinated mice by MTT assay. The E7-specific cytotoxic T cells (CTL) were analyzed by lymphocyte proliferation and lactate dehydrogenates (LDH) release assays. IFN-γ, IL-4 and TNF-α secretion in splenocyte cultures as well as vascular endothelial growth factor (VEGF) and IL-10 in the tumor microenvironment were assayed by ELISA. Our results demonstrated that subcutaneous administration of C57BL/6 mice with a DNA vaccine adjuvanted with MLT dose-dependently and significantly induced strong HPV16 E7-specific CD8+ cytotoxicity and IFN-γ and TNF-α responses capable of reducing HPV-16 E7-expressing tumor volume. A significantly higher level of E7-specific T-cell proliferation was also found in the adjuvanted vaccine group. Furthermore, tumor growth was significantly inhibited when the DNA vaccine was combined with MLT and the survival time of TC-1 tumor bearing mice was also significantly prolonged. In vivo studies further demonstrated that MLT decreased the accumulation of IL-10 and VEGF in the tumor microenvironment of vaccinated mice. These data indicate that melatonin as an adjuvant augmented the cancer vaccine efficiency against HPV-associated tumors in a dose dependent manner.

Rapamycin Augments Immunomodulatory Properties of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis.

The immunomodulatory and anti-inflammatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) have been considered as an appropriate candidate for treatment of autoimmune diseases. Previous studies have revealed that treatment with BM-MSCs may modulate immune responses and alleviate the symptoms in experimental autoimmune encephalomyelitis (EAE) mice, an animal model of multiple sclerosis. Therefore, the present study was designed to examine immunomodulatory effects of BM-MSCs in the treatment of myelin oligodendrocyte glycoprotein (MOG) 35-55-induced EAE in C57BL/6 mice. MSCs were obtained from the bone marrow of C57BL mice, cultured with DMEM/F12, and characterized with flow cytometry for the presence of cell surface markers for BM-MSCs. Following three passages, BM-MSCs were injected intraperitoneally into EAE mice alone or in combination with rapamycin. Immunological and histopathological effects of BM-MSCs and addition of rapamycin to BM-MSCs were evaluated. The results demonstrated that adding rapamycin to BM-MSCs transplantation in EAE mice significantly reduced inflammation infiltration and demyelination, enhanced the immunomodulatory functions, and inhibited progress of neurological impairments compared to BM-MSC transplantation and control groups. The immunological effects of rapamycin and BM-MSC treatments were associated with the inhibition of the Ag-specific lymphocyte proliferation, CD8+ cytolytic activity, and the Th1-type cytokine (gamma-interferon (IFN-γ)) and the increase of Th-2 cytokine (interleukin-4 (IL-4) and IL-10) production. Addition of rapamycin to BM-MSCs was able to ameliorate neurological deficits and provide neuroprotective effects in EAE. This suggests the potential of rapamycin and BM-MSC combined therapy to play neuroprotective roles in the treatment of neuroinflammatory disorders.

Melatonin reverses H-89 induced spatial memory deficit: Involvement of oxidative stress and mitochondrial function.

Oxidative stress and mitochondrial dysfunction play indispensable role in memory and learning impairment. Growing evidences have shed light on anti-oxidative role for melatonin in memory deficit. We have previously reported that inhibition of protein kinase A by H-89 can induce memory impairment. Here, we investigated the effect of melatonin on H-89 induced spatial memory deficit and pursued their interactive consequences on oxidative stress and mitochondrial function in Morris Water Maze model. Rats received melatonin (50 and 100µg/kg/side) and H-89(10µM) intra-hippocampally 30min before each day of training. Animals were trained for 4 consecutive days, each containing one block from four trials. Oxidative stress indices, including thiobarbituric acid (TBARS), reactive oxygen species (ROS), thiol groups, and ferric reducing antioxidant power (FRAP) were assessed using spectrophotometer. Mitochondrial function was evaluated through measuring ROS production, mitochondrial membrane potential (MMP), swelling, outer membrane damage, and cytochrome c release. As expected from our previous report, H-89 remarkably impaired memory by increasing the escape latency and traveled distance. Intriguingly, H-89 significantly augmented TBARS and ROS levels, caused mitochondrial ROS production, swelling, outer membrane damage, and cytochrome c release. Moreover, H-89 lowered thiol, FRAP, and MMP values. Intriguingly, melatonin pre-treatment not only effectively hampered H-89-mediated spatial memory deficit at both doses, but also reversed the H-89 effects on mitochondrial and biochemical indices upon higher dose. Collectively, these findings highlight a protective role for melatonin against H-89-induced memory impairment and indicate that melatonin may play a therapeutic role in the treatment of oxidative- related neurodegenerative disorders.

Protective effects of salicylate on PKA inhibitor (H-89)-induced spatial memory deficit via lessening autophagy and apoptosis in rats.

In this study, the effects of salicylate on spatial learning and memory, through its effects on autophagy and apoptosis, were evaluated in the presence of the PKA inhibitor H-89. Adult male Wistar rats were divided into experimental groups as follows: salicylate (30, 50, 100µg/0.5µl/side, intra-hippocampal; 400mg/kg, intra-peritoneal), donepezil (1mg/kg as a positive control for behavioral effects of salicylate), H-89 (1µl/side of 5 or 20µM), H-89 plus salicylate and H-89 plus donepezil. The Morris water maze test was used for evaluation of spatial learning and memory. The levels of different apoptotic and autophagic biomarkers were evaluated using the western blot technique. Salicylate (100µg/0.5µl/side) significantly reduced the escape latency on training days, increased the percentage of time spent in the target quadrant during the probe trial and reversed the inhibitory effects of H-89 during the process of spatial learning and memory. The behavioral efficacy of salicylate was comparable to that of donepezil. In addition, salicylate significantly decreased the levels of apoptotic proteins, Bax and caspase 3, and increased the Bcl2 levels in all groups. Furthermore, the levels of LC3II and Atg7 were decreased by salicylate. Our study revealed that both systemic and direct intra-hippocampal administration of salicylate can facilitate the spatial learning and memory. Additionally, intra-hippocampal administration of salicylate can reduce apoptotic and autophagic proteins. The antioxidant activity of salicylate might lead to increased pCREB via stimulation of signaling pathways, resulting in reduction of H-89-induced apoptosis and autophagy.

The Effect of Melatonin on Behavioral, Molecular, and Histopathological Changes in Cuprizone Model of Demyelination.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. The protective effects of melatonin (MLT) on various neurodegenerative diseases, including MS, have been suggested. In the present study, we examined the effect of MLT on demyelination, apoptosis, inflammation, and behavioral dysfunctions in the cuprizone toxic model of demyelination. C57BL/6J mice were fed a chaw containing 0.2 % cuprizone for 5 weeks and received two doses of MLT (50 and 100 mg/kg) intraperitoneally for the last 7 days of cuprizone diet. Administration of MLT improved motor behavior deficits induced by cuprizone diet. MLT dose-dependently decreased the mean number of apoptotic cells via decreasing caspase-3 and Bax as well as increasing Bcl-2 levels. In addition, MLT significantly enhanced nuclear factor-κB activation and decreased heme oxygenase-1 level. However, MLT had no effect on interleukin-6 and myelin protein production. Our data revealed that MLT improved neurological deficits and enhanced cell survival but was not able to initiate myelin production in the cuprizone model of demyelination. These findings may be important for the design of potential MLT therapy in demyelinating disorders, such as MS.

Protective Effect of a cAMP Analogue on Behavioral Deficits and Neuropathological Changes in Cuprizone Model of Demyelination.

Multiple sclerosis (MS) is an inflammatory demyelinating disease that leads to neuronal cell loss. Cyclic AMP and its analogs are well known to decrease inflammation and apoptosis. In the present study, we examined the effects of bucladesine, a cell-permeable analogue of cyclic adenosine monophosphate (cAMP), on myelin proteins (PLP, PMP-22), inflammation, and apoptotic, as well as anti-apoptotic factors in cuprizone model of demyelination. C57BL/6J mice were fed with chow containing 0.2% copper chelator cuprizone or vehicle by daily oral gavage for 5 weeks to induce reversible demyelination predominantly of the corpus callosum. Bucladesine was administered intraperitoneally at different doses (0.24, 0.48, or 0.7 µg/kg body weight) during the last 7 days of 5-week cuprizone treatment. Bucladesine exhibited a protective effect on myelination. Furthermore, bucladesine significantly decreased the production of interleukin-6 pro-inflammatory mediator as well as nuclear factor-κB activation and reduced the mean number of apoptotic cells compared to cuprizone-treated mice. Bucladesine also decreased production of caspase-3 as well as Bax and increased Bcl-2 levels. Our data revealed that enhancement of intracellular cAMP prevents demyelination and plays anti-inflammatory and anti-apoptotic properties in mice cuprizone model of demyelination. This suggests the modulation of intracellular cAMP as a potential target for treatment of MS.

Interactive effects of a protein kinase AII inhibitor and testosterone on spatial learning in the Morris water maze.

Neurohormones such as testosterone (TE) are important in modulation of learning and memory. In the present study, we investigated the interactive effects of pre-training bilateral intra-hippocampal infusions of testosterone and H-89, a selective PKAII inhibitor, on spatial acquisition in the Morris water maze (MWM). Different doses of TE (20, 40 and 80 µg/side) and H-89 (5 and 10 µM/side) were administered 30 min before start of the training each day. Control animals received bilateral intra-hippocampal infusions of DMSO as vehicle for TE and H-89. Animals were trained for 4 days and each day included one block of four trials. The results of this study showed that bilateral infusion of TE (40 and 80 µg/side) or H-89 (10 µM/side) impaired spatial learning as indicated by significant increases in escape latency and traveled distance compared to the control group. Although pre-training bilateral infusions of a low concentration of either TE (20 µg/side) or H-89 (5 µM/side) into the CA1 region of the hippocampus did not affect learning capabilities, but the combination of the low doses of the drugs led to significant deficits in spatial acquisition. Overall, our data suggest that spatial acquisition was affected by PKAII inhibition or TE administration. Moreover, when co-administered, these drugs had a negative synergistic impact on acquisition.