Long-term effects of myo-inositol on traumatic brain injury: Epigenomic and transcriptomic studies.

Background and purpose: Traumatic brain injury (TBI) and its consequences remain great challenges for neurology. Consequences of TBI are associated with various alterations in the brain but little is known about long-term changes of epigenetic DNA methylation patterns. Moreover, nothing is known about potential treatments that can alter these epigenetic changes in beneficial ways. Therefore, we have examined myo-inositol (MI), which has positive effects on several pathological conditions. Methods: TBI was induced in mice by controlled cortical impact (CCI). One group of CCI animals received saline injections for two months (TBI+SAL), another CCI group received MI treatment (TBI+MI) for the same period and one group served as a sham-operated control. Mice were sacrificed 4 months after CCI and changes in DNA methylome and transcriptomes were examined. Results: For the first time we: (i) provide comprehensive map of long-term DNA methylation changes after CCI in the hippocampus; (ii) identify differences by methylation sites between the groups; (iii) characterize transcriptome changes; (iv) provide association between DNA methylation sites and gene expression. MI treatment is linked with upregulation of genes covering 33 biological processes, involved in immune response and inflammation. In support of these findings, we have shown that expression of BATF2, a transcription factor involved in immune-regulatory networks, is upregulated in the hippocampus of the TBI+MI group where the BATF2 gene is demethylated. Conclusion: TBI is followed by long-term epigenetic and transcriptomic changes in hippocampus. MI treatment has a significant effect on these processes by modulation of immune response and biological pathways of inflammation.

Myo-Inositol Limits Kainic Acid-Induced Epileptogenesis in Rats.

Epilepsy is a severe neurological disease characterized by spontaneous recurrent seizures (SRS). A complex pathophysiological process referred to as epileptogenesis transforms a normal brain into an epileptic one. Prevention of epileptogenesis is a subject of intensive research. Currently, there are no clinically approved drugs that can act as preventive medication. Our previous studies have revealed highly promising antiepileptogenic properties of a compound-myo-inositol (MI) and the present research broadens previous results and demonstrates the long-term disease-modifying effect of this drug, as well as the amelioration of cognitive comorbidities. For the first time, we show that long-term treatment with MI: (i) decreases the frequency and duration of electrographic SRS in the hippocampus; (ii) has an ameliorating effect on spatial learning and memory deficit associated with epileptogenesis, and (iii) attenuates cell loss in the hippocampus. MI treatment also alters the expression of the glial fibrillary acidic protein, LRRC8A subunit of volume-regulated anion channels, and protein tyrosine phosphatase receptor type R, all expected to counteract the epileptogenesis. All these effects are still present even 4 weeks after MI treatment ceased. This suggests that MI may exert multiple actions on various epileptogenesis-associated changes in the brain and, therefore, could be considered as a candidate target for prevention of epileptogenesis.

Long-Term Effects of Myoinositol on Behavioural Seizures and Biochemical Changes Evoked by Kainic Acid Induced Epileptogenesis.

Epilepsy is one of the most devastating neurological diseases and despite significant efforts there is no cure available. Occurrence of spontaneous seizures in epilepsy is preceded by numerous functional and structural pathophysiological reorganizations in the brain-a process called epileptogenesis. Treatment strategies targeting this process may be efficient for preventing spontaneous recurrent seizures (SRS) in epilepsy, or for modification of disease progression. We have previously shown that (i) myoinositol (MI) pretreatment significantly decreases severity of acute seizures (status epilepticus: SE) induced by kainic acid (KA) in experimental animals and (ii) that daily post-SE administration of MI for 4 weeks prevents certain biochemical changes triggered by SE. However it was not established whether such MI treatment also exerts long-term effects on the frequency of SRS. In the present study we have shown that, in KA-induced post-SE epilepsy model in rats, MI treatment for 28 days reduces frequency and duration of behavioural SRS not only during the treatment, but also after its termination for the following 4 weeks. Moreover, MI has significant effects on molecular changes in the hippocampus, including mi-RNA expression spectrum, as well as mRNA levels of sodium-MI transporter and LRRC8A subunit of the volume regulated anionic channel. Taken together, these data suggest that molecular changes induced by MI treatment may counteract epileptogenesis. Thus, here we provide data indicating antiepileptogenic properties of MI, which further supports the idea of developing new antiepileptogenic and disease modifying drug that targets MI system.

Myoinositol Attenuates the Cell Loss and Biochemical Changes Induced by Kainic Acid Status Epilepticus.

Identification of compounds preventing or modifying the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action are of great importance. We have previously shown that myoinositol (MI) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (KA) induced status epilepticus (SE). However in these studies we have not detected any effects of MI on the first day after SE. In the present study we broadened our research and focused on other molecular and morphological changes at the early stages of SE induced by KA and effects of MI treatment on these changes. The increase in the amount of voltage-dependent anionic channel-1 (VDAC-1), cofilin, and caspase-3 activity was observed in the hippocampus of KA treated rats. Administration of MI 4 hours later after KA treatment abolishes these changes, whereas diazepam treatment by the same time schedule has no significant influence. The number of neuronal cells in CA1 and CA3 subfields of hippocampus is decreased after KA induced SE and MI posttreatment significantly attenuates this reduction. No significant changes are observed in the neocortex. Obtained results indicate that MI posttreatment after KA induced SE could successfully target the biochemical processes involved in apoptosis, reduces cell loss, and can be successfully used in the future for translational research.

Posttraumatic seizures and epilepsy in adult rats after controlled cortical impact.

Posttraumatic epilepsy (PTE) has been modeled with different techniques of experimental traumatic brain injury (TBI) using mice and rats at various ages. We hypothesized that the technique of controlled cortical impact (CCI) could be used to establish a model of PTE in young adult rats. A total of 156 male Sprague-Dawley rats of 2-3 months of age (128 CCI-injured and 28 controls) was used for monitoring and/or anatomical studies. Provoked class 3-5 seizures were recorded by video monitoring in 7/57 (12.3%) animals in the week immediately following CCI of the right parietal cortex; none of the 7 animals demonstrated subsequent spontaneous convulsive seizures. Monitoring with video and/or video-EEG was performed on 128 animals at various time points 8-619 days beyond one week following CCI during which 26 (20.3%) demonstrated nonconvulsive or convulsive epileptic seizures. Nonconvulsive epileptic seizures of >10s were demonstrated in 7/40 (17.5%) animals implanted with 2 or 3 depth electrodes and usually characterized by an initial change in behavior (head raising or animal alerting) followed by motor arrest during an ictal discharge that consisted of high-amplitude spikes or spike-waves with frequencies ranging between 1 and 2Hz class 3-5 epileptic seizures were recorded by video monitoring in 17/88 (19%) and by video-EEG in 2/40 (5%) CCI-injured animals. Ninety of 156 (58%) animals (79 CCI-injured, 13 controls) underwent transcardial perfusion for gross and microscopic studies. CCI caused severe brain tissue loss and cavitation of the ipsilateral cerebral hemisphere associated with cell loss in the hippocampal CA1 and CA3 regions, hilus, and dentate granule cells, and thalamus. All Timm-stained CCI-injured brains demonstrated ipsilateral hippocampal mossy fiber sprouting in the inner molecular layer. These results indicate that the CCI model of TBI in adult rats can be used to study the structure-function relationships that underlie epileptogenesis and PTE.

Age-dependent loss of parvalbumin-expressing hippocampal interneurons in mice deficient in CHL1, a mental retardation and schizophrenia susceptibility gene.

In humans, deletions/mutations in the CHL1/CALL gene are associated with mental retardation and schizophrenia. Juvenile CHL1-deficient (CHL1(-/-) ) mice have been shown to display abnormally high numbers of parvalbumin-expressing (PV(+) ) hippocampal interneurons and, as adults, display behavioral traits observed in neuropsychiatric disorders. Here, we addressed the question whether inhibitory interneurons and synaptic plasticity in the CHL1(-/-) mouse are affected during brain maturation and in adulthood. We found that hippocampal, but not neocortical, PV(+) interneurons were reduced with age in CHL1(-/-) mice, from a surplus of +27% at 1 month to a deficit of -20% in adulthood compared with wild-type littermates. This loss occurred during brain maturation, correlating with microgliosis and enhanced interleukin-6 expression. In parallel with the loss of PV(+) interneurons, the inhibitory input to adult CA1 pyramidal cells was reduced and a deficit in short- and long-term potentiation developed at CA3-CA1 excitatory synapses between 2 and 9 months of age in CHL1(-/-) mice. This deficit could be abrogated by a GABAA receptor agonist. We propose that region-specific aberrant GABAergic synaptic connectivity resulting from the mutation and a subsequently enhanced synaptic elimination during brain maturation lead to microgliosis, increase in pro-inflammatory cytokine levels, loss of interneurons, and impaired synaptic plasticity. Close homolog of L1-deficient (CHL1(-/-) ) mice have abnormally high numbers of parvalbumin (PV)-expressing hippocampal interneurons in juvenile animals, but in adult animals a loss of these cells is observed. This loss correlates with an increased density of microglia (M), enhanced interleukin-6 (IL6) production and a deficit in short- and long-term potentiation at CA3-CA1 excitatory synapses. Furthermore, adult CHL1(-/-) mice display behavioral traits similar to those observed in neuropsychiatric disorders of humans.

Myo-inositol treatment and GABA-A receptor subunit changes after kainate-induced status epilepticus.

Identification of compounds preventing the biochemical changes that underlie the epileptogenesis process is of great importance. We have previously shown that myo-Inositol (MI) daily treatment prevents certain biochemical changes that are triggered by kainic acid (KA)-induced status epilepticus (SE). The aim of the current work was to study the further influence of MI treatment on the biochemical changes of epileptogenesis and focus on changes in the hippocampus and neocortex of rats for the following GABA-A receptor subunits: α1, α4, γ2, and δ. After SE, one group of rats was treated with saline, while the second group was treated with MI. Control groups that were not treated by the convulsant received either saline or MI administration. 28-30 h after the experiment, a decrease in the amount of the α1 subunit was revealed in the hippocampus and MI had no significant influence on it. On the 28th day of the experiment, the amount of α1 was increased in both the KA- and KA + MI-treated groups. The α4 and γ2 subunits were strongly reduced in the hippocampus of KA-treated animals, but MI significantly halted this reduction. The effects of MI on α4 and γ2 subunit changes were significantly different between hippocampus and neocortex. On the twenty-eighth day after SE, a decrease in the amount of α1 was found in the neocortex, but MI treatment had no effect on it. The obtained results indicate that MI treatment interferes with some of the biochemical processes of epileptogenesis.

Alterations of GABA(A) and glutamate receptor subunits and heat shock protein in rat hippocampus following traumatic brain injury and in posttraumatic epilepsy.

Traumatic brain injury (TBI) can result in the development of posttraumatic epilepsy (PTE). Recently, we reported differential alterations in tonic and phasic GABA(A) receptor (GABA(A)R) currents in hippocampal dentate granule cells 90 days after controlled cortical impact (CCI) (Mtchedlishvili et al., 2010). In the present study, we investigated long-term changes in the protein expression of GABA(A)R α1, α4, γ2, and δ subunits, NMDA (NR2B) and AMPA (GluR1) receptor subunits, and heat shock proteins (HSP70 and HSP90) in the hippocampus of Sprague-Dawley rats evaluated by Western blotting in controls, CCI-injured animals without PTE (CCI group), and CCI-injured animals with PTE (PTE group). No differences were found among all three groups for α1 and α4 subunits. Significant reduction of γ2 protein was observed in the PTE group compared to control. CCI caused a 194% and 127% increase of δ protein in the CCI group compared to control (p<0.0001), and PTE (p<0.0001) groups, respectively. NR2B protein was increased in CCI and PTE groups compared to control (p=0.0001, and p=0.011, respectively). GluR1 protein was significantly decreased in CCI and PTE groups compared to control (p=0.003, and p=0.001, respectively), and in the PTE group compared to the CCI group (p=0.036). HSP70 was increased in CCI and PTE groups compared to control (p=0.014, and p=0.005, respectively); no changes were found in HSP90 expression. These results provide for the first time evidence of long-term alterations of GABA(A) and glutamate receptor subunits and a HSP following CCI.

Increase of GABAA receptor-mediated tonic inhibition in dentate granule cells after traumatic brain injury.

Traumatic brain injury (TBI) can result in altered inhibitory neurotransmission, hippocampal dysfunction, and cognitive impairments. GABAergic spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) and tonic (extrasynaptic) whole cell currents were recorded in control rat hippocampal dentate granule cells (DGCs) and at 90days after controlled cortical impact (CCI). At 34 degrees C, in CCI DGCs, sIPSC frequency and amplitude were unchanged, whereas mIPSC frequency was decreased (3.10+/-0.84Hz, n=16, and 2.44+/-0.67Hz, n=7, p<0.05). At 23 degrees C, 300nM diazepam increased peak amplitude of mIPSCs in control and CCI DGCs, but the increase was 20% higher in control (26.81+/-2.2pA and 42.60+/-1.22pA, n=9, p=0.031) compared to CCI DGCs (33.46+/-2.98pA and 46.13+/-1.09pA, n=10, p=0.047). At 34 degrees C, diazepam did not prolong decay time constants (6.59+/-0.12ms and 6.62+/-0.98ms, n=9, p=0.12), the latter suggesting that CCI resulted in benzodiazepine-insensitive pharmacology in synaptic GABA(A) receptors (GABA(A)Rs). In CCI DGCs, peak amplitude of mIPSCs was inhibited by 100microM furosemide (51.30+/-0.80pA at baseline and 43.50+/-5.30pA after furosemide, n=5, p<0.001), a noncompetitive antagonist of GABA(A)Rs with an enhanced affinity to alpha4 subunit-containing receptors. Potentiation of tonic current by the GABA(A)R delta subunit-preferring competitive agonist THIP (1 and 3microM) was increased in CCI DGCs (47% and 198%) compared to control DGCs (13% and 162%), suggesting the presence of larger tonic current in CCI DGCs; THIP (1microM) had no effect on mIPSCs. Taken together, these results demonstrate alterations in synaptic and extrasynaptic GABA(A)Rs in DGCs following CCI.

Myo-inositol treatment prevents biochemical changes triggered by kainate-induced status epilepticus.

Identification of the compounds preventing the biochemical changes underlying the epileptogenesis process is of great importance. We have previously shown that myo-inositol (MI) administration reduces kainic acid (KA) induced seizure scores. MI treatment effects on biochemical changes triggered by KA induced status epilepticus (SE) were investigated in the present study. After SE one group of rats was treated with saline, whereas the second group with MI. Control groups received either saline or MI administration. Changes in the amounts of following proteins were studied in the hippocampus and neocortex of rats: GLUR1 subunit of glutamate receptors, calcium/calmodulin-dependent protein kinase II (CaMKII), and heat shock protein 90. No changes were found 28-30h after experiments. However on 28th day of experiment the amounts of GLUR1 and CaMKII were strongly reduced in the hippocampus of KA treated animals but MI significantly halted this reduction. Obtained results indicate anti-epileptogenic features of MI on biochemical level.

Reduced reactivity to novelty, impaired social behavior, and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule CHL1.

The neural cell adhesion molecule CHL1 is implicated in neural development in the mouse and has been related to psychiatric disorders in humans. Here we report that mice constitutively deficient for CHL1 display reduced reactivity to environmental stimuli and reduced expression of social behaviors, whereas cognitive, motor and olfactory functions are normal. Basal synaptic transmission and plasticity in seven major excitatory connections in the hippocampus were analyzed to test whether dysfunctions in this brain region, which controls complex behaviors, correlate with the behavioral alterations of CHL1 deficient mice. We found that basal synaptic transmission in lateral and medial perforant path projections to the dentate gyrus is elevated in CHL1-deficient mice. Taking in consideration the function of these synapses in processing information from cortical areas, we hypothesize that constitutive ablation of CHL1 leads to reduced capability to react to external stimuli due to dysfunctions in the dentate gyrus.

Enhanced perisomatic inhibition and impaired long-term potentiation in the CA1 region of juvenile CHL1-deficient mice.

The cell adhesion molecule, CHL1, like its close homologue L1, is important for normal brain development and function. In this study, we analysed the functional role of CHL1 in synaptic transmission in the CA1 region of the hippocampus using juvenile CHL1-deficient (CHL1-/-) and wild-type (CHL1+/+) mice. Inhibitory postsynaptic currents evoked in pyramidal cells by minimal stimulation of perisomatically projecting interneurons were increased in CHL1-/- mice compared with wild-type littermates. Also, long-term potentiation (LTP) at CA3-CA1 excitatory synapses was reduced under physiological conditions in CHL1-/- mice. This abnormality was abolished by application of a GABAA receptor antagonist, suggesting that enhanced inhibition is the cause of LTP impairment. Quantitative ultrastructural and immunohistochemical analyses revealed aberrations possibly related to the abnormally high inhibition observed in CHL1-/- mice. The length and linear density of active zones in symmetric synapses on pyramidal cell bodies, as well as number of perisomatic puncta containing inhibitory axonal markers were increased. Density and total number of parvalbumin-positive interneurons was also abnormally high. These observations and the finding that CA1 interneurons express CHL1 protein indicate that CHL1 is important for regulation of inhibitory synaptic transmission and interneuron populations in the postnatal brain. The observed enhancement of inhibitory transmission in CHL1-/- mice is in contrast to the previous finding of reduced inhibition in L1 deficient mice and indicates different functions of these two closely related molecules.

Effects of phencyclidines on signal transfer from the entorhinal cortex to the hippocampus in rats.

The information transfer from the superficial layers of the entorhinal cortex (EC) to the hippocampus is regulated in a frequency dependent manner. Phencyclidine and related compounds such as MK-801 produce psychotic symptoms that closely resemble schizophrenia. We studied the effects of systemic administration of MK-801 on the signal transfer from the EC layer III to the hippocampal area CA1. High frequency (above 10 Hz) activation of the bi-synaptic entorhinal input in control animals results in a strong suppression of the field potentials in the stratum lacunosum-moleculare of the area CA1. In contrast, in MK-801 pretreated rats the field response was less reduced. The field potential responses evoked in these two groups of animals by high-frequency activation of the monosynaptic input were similar suggesting selective alterations in layer III of the medial EC. We suggest, that MK-801 causes disinhibition of layer III projection cells and, therefore, may cause strong, pathological activation of direct layer III-CA1 pathway.