Histopathological and Biochemical Assessment of Neuroprotective Effects of Sodium Valproate and Lutein on the Pilocarpine Albino Rat Model of Epilepsy

Epilepsy is one of the most frequent neurological disorders characterized by an enduring predisposition to generate epileptic seizures. Oxidative stress is believed to directly participate in the pathways of neurodegenerations leading to epilepsy. Approximately, one-third of the epileptic patients who suffer from seizures do not receive effective medical treatment. Sodium valproate (SVP) is a commonly used antiepileptic drug (AED); however, it has toxic effects. Lutein (L), a carotenoid, has potent antioxidant and anti-inflammatory properties. The aim of this study was to determine the neuroprotective effect of sodium valproate (SVP) and lutein (L) in a rat model of pilocarpine- (PLC-) induced epilepsy. To achieve this aim, fifty rats were randomly divided into five groups. Group I: control, group II: received PLC (400 mg/kg intraperitoneally), group III: received PLC + SVP (500 mg/kg orally), group IV: received PLC + SVP + L (100 mg/kg orally), and group V: received (PLC + L). Racine Scale (RC) and latency period to onset seizure were calculated. After eight weeks, the hippocampus rotarod performance and histological investigations were performed. Oxidative stress was investigated in hippocampal homogenates. Results revealed that SVP and L, given alone or in combination, reduced the RC significantly, a significant delay in latency to PLC-kindling onset, and improved rotarod performance of rats compared with the PLC group. Moreover, L was associated with a reduction of oxidative stress in hippocampal homogenate, a significant decrease in serum tumor necrosis factor-alpha (TNF-α) level, and inhibition of cerebral injury and displayed antiepileptic properties in the PLC-induced epileptic rat model. Data obtained from the current research elucidated the prominent neuroprotective, antioxidant, and anti-inflammatory activities of lutein in this model. In conclusion, lutein cotreatment with AEDs is likely to be a promising strategy to improve treatment efficacy in patients suffering from epilepsy.

Repurposing of Dapsone as Neuroprotective Agent on Cuprizone-induced Demyelination via Targeting Nrf2 and NF-kB in C57BL/6 Mice

Multiple Sclerosis (MS) is an inflammatory disorder wherein the myelin of nerve cells in central nervous system is damaged. Fatigue, lack of coordination, blurred vision and mental problems are some of the symptoms of MS. In the current study, we assessed the performance of Dapsone (DAP) on improvement of behavioral dysfunction and preservation of myelin in the cuprizone (CPZ) induced demyelination model via targeting Nrf2 and IKB. MS was induced in C57BL/6 mice through diet supplementation of CPZ (0.2%) for 6 weeks and DAP (12.5 mg/kg/day; i.p.) was administered once daily for the last 2 weeks of treatment. Behavioral tests (pole test and rotarod performance test), LFB and H&E staining and Immunohistochemistry (IHC) staining of p-Nrf2 and p-IKB were performed to evaluate locomotor coordination, observe the area of demyelination in corpus callosum respectively. Furthermore, superoxide dismutase (SOD), and nitrite were measured. Based on the results of the weight of mice, it was shown that the weight of the groups receiving CPZ decreased compared to the control group (P < 0.001), while administration of dapsone increased the weight (P < 0.001). Pole test showed that CPZ increased latency time to fall (P <0.01) but the latency to reach the floor in DAP-CPZ group was significantly shorter than the CPZ group (P <0.001). Rotarod performance test showed the effect of CPZ in reducing fall time in CPZ group (P <0.01) however treatment with DAP significantly increased fall time (P < 0.001). In LFB staining increased demyelination was seen in the CPZ group and decreased demyelination was observed in the DAP group. IHC staining results of p-Nrf2 and p-IKB showed that CPZ significantly decreased p-Nrf2 and elevated p-IKB levels compared with the control group (P <0.001), but in DAP -treated groups markedly modified these changes (P < 0.001). CPZ feeding led to increase of brain nitrite levels and reduced SOD activity (P <0.05) but in DAP-treated considerably reversed CPZ-induced changes in nitrite and SOD activity level (P <0.001). These data support the suggestion that the beneficial properties of dapsone on the CPZ-induced demyelination are mediated by targeting Nrf2 and NF-kB pathways.

Electroacupuncture may alleviate behavioral defects via modulation of gut microbiota in a mouse model of Parkinson’s disease

Objective: Parkinson’s disease (PD) is a chronic neurodegenerative disease involving non-motor symptoms, of which gastrointestinal disorders are the most common. In light of recent results, intestinal dysfunction may be involved in the pathogenesis of PD. Electroacupuncture (EA) has shown potential effects, although the underlying mechanism remains mostly unknown. We speculated that EA could relieve the behavioral defects of PD, and that this effect would be associated with modulation of the gut microbiota. Methods: Mice were randomly divided into three groups: control, PD + MA (manual acupuncture), and PD + EA. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was used to establish the mouse model of PD. Rotarod performance tests, open field tests, and pole tests were carried out to assess motor deficiencies. Immunohistochemistry was conducted to examine the survival of dopaminergic neurons. 16S ribosomal RNA (rRNA) gene sequencing was applied to investigate the alterations of the gut microbiome. Quantitative real-time polymerase chain reaction (PCR) was performed to characterize the messenger RNA (mRNA) levels of pro-inflammatory and anti-inflammatory cytokines. Results: We found that EA was able to alleviate the behavioral defects in the rotarod performance test and pole test, and partially rescue the significant loss of dopaminergic neurons in the substantia nigra (SN) chemically induced by MPTP in mice. Moreover, the PD + MA mice showed a tendency toward decreased intestinal microbial alpha diversity, while EA significantly reversed it. The abundance of Erysipelotrichaceae was significantly increased in PD + MA mice, and the alteration was also reversed by EA. In addition, the pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were substantially increased in the SN of PD + MA mice, an effect that was reversed by EA. Conclusion: These results suggest that EA may alleviate behavioral defects via modulation of gut microbiota and suppression of inflammation in the SN of mice with PD, which provides new insights into the pathogenesis of PD and its treatment.

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead ( RIPK1D138N/D138N) mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL ( Mlkl-/-) had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.

Subarachnoid hemorrhage leads to early and persistent functional connectivity and behavioral changes in mice

Aneurysmal subarachnoid hemorrhage (SAH) leads to significant long-term cognitive deficits, which can be associated with alterations in resting state functional connectivity (RSFC). However, modalities such as fMRI—which is commonly used to assess RSFC in humans—have practical limitations in small animals. Therefore, we used non-invasive optical intrinsic signal imaging to determine the effect of SAH on RSFC in mice up to three months after prechiasmatic blood injection. We assessed Morris water maze (MWM), open field test (OFT), Y-maze, and rotarod performance from approximately two weeks to three months after SAH. Compared to sham, we found that SAH reduced motor, retrosplenial, and visual seed-based connectivity indices. These deficits persisted in retrosplenial and visual cortex seeds at three months. Seed-to-seed analysis confirmed early attenuation of correlation coefficients in SAH mice, which persisted in predominantly posterior network connections at later time points. Seed-independent global and interhemispheric indices of connectivity revealed decreased correlations following SAH for at least one month. SAH led to MWM hidden platform and OFT deficits at two weeks, and Y-maze deficits for at least three months, without altering rotarod performance. In conclusion, experimental SAH leads to early and persistent alterations both in hemodynamically derived measures of RSFC and in cognitive performance.

Low acetylcholine during early sleep is important for motor memory consolidation

The synaptic homeostasis theory of sleep proposes that low neurotransmitter activity in sleep optimizes memory consolidation. We tested this theory by asking whether increasing acetylcholine levels during early sleep would weaken motor memory consolidation. We trained separate groups of adult mice on the rotarod walking task and the single pellet reaching task, and after training, administered physostigmine, an acetylcholinesterase inhibitor, to increase cholinergic tone in subsequent sleep. Post-sleep testing showed that physostigmine impaired motor skill acquisition of both tasks. Home-cage video monitoring and electrophysiology revealed that physostigmine disrupted sleep structure, delayed non-rapid-eye-movement sleep onset, and reduced slow-wave power in the hippocampus and cortex. Additional experiments showed that: (1) the impaired performance associated with physostigmine was not due to its effects on sleep structure, as 1 h of sleep deprivation after training did not impair rotarod performance, (2) a reduction in cholinergic tone by inactivation of cholinergic neurons during early sleep did not affect rotarod performance, and (3) stimulating or blocking muscarinic and nicotinic acetylcholine receptors did not impair rotarod performance. Taken together, the experiments suggest that the increased slow wave activity and inactivation of both muscarinic and nicotinic receptors during early sleep due to reduced acetylcholine contribute to motor memory consolidation.

Subarachnoid hemorrhage leads to early and persistent functional connectivity and behavioral changes in mice

Aneurysmal subarachnoid hemorrhage (SAH) leads to significant long-term cognitive deficits. Studies in survivors of SAH show an association between persistent cognitive deficits and alterations in resting state functional connectivity (RSFC). However, modalities commonly used to assess RSFC in humans, such as fMRI, have practical limitations in small animals. Therefore, we used non-invasive functional optical intrinsic signal imaging to determine the effect of SAH on measures of RSFC in mice at early (day 4), intermediate (1 month), and late (3 months) time points after prechiasmatic arterial blood injection. We assessed Morris water maze, open field test, Y-maze, and rotarod performance from approximately 2 weeks to 3 months after SAH induction. We found qualitative and quantitative differences in seed-based connectivity maps between sham and SAH mice. SAH reduced motor, retrosplenial and visual seed-based connectivity indices, which persisted in retrosplenial and visual cortex seeds at 3 months. Seed-to-seed connectivity analysis confirmed attenuation of correlation coefficients in SAH mice, which persisted in predominantly posterior network connections at later time points. Seed-independent global and interhemispheric indices of connectivity revealed decreased correlations following SAH for at least 1 month. SAH led to Morris water maze hidden platform and open field deficits at 2 weeks, and Y-maze deficits for at least 3 months, without altering rotarod performance. In conclusion, experimental SAH leads to early and persistent alterations both in hemodynamically-derived measures of RSFC and in cognitive performance.

Effects of intraplantar administration of Complete Freund’s Adjuvant (CFA) on rotarod performance in mice

Background and aims Preclinical animal models are crucial to study pain mechanisms and assess antinociceptive effects of medications. One major problem with current animal behavioral models is their lack of face validity with human nociception and the vulnerability for false-positive results. Here, we evaluated the usefulness of rotarod as a new way to assess inflammatory nociception in rodents. Methods Adult male mice were injected with saline or Complete Freund’s Adjuvant (CFA) in the left hindpaws. Mechanical allodynia and rotarod performance were evaluated before and after the administration of CFA. Mechanical allodynia was measured using von Frey filaments. Long-term effect of CFA on rotarod performance was also assessed for 2 weeks. Results Our results showed that CFA administration decreased pain threshold and increased sensitivity to von Frey filaments compared to control group. In rotarod experiments, the starting speed of the rod rotation started at four RPM, and accelerated until it reached 40 RPM in 5 min. Rotarod performance was enhanced from day to day in the control group. However, rotarod performance in CFA group was attenuated after CFA administration, which was significant after 24 h compared to vehicle. This attenuation was blocked by ibuprofen. Haloperidol administration (positive control) produced similar results to CFA administration. CFA did not produce significant attenuation of rotarod performance after 1 week post-injection. Conclusions Collectively, our findings could encourage the use of rotarod assay to measure acute (but not chronic) inflammatory nociception as a useful tool in rodents.

Low acetylcholine during early sleep is important for motor memory consolidation

The synaptic homeostasis theory of sleep proposes that low neurotransmitter activity in sleep is optimal for memory consolidation. We tested this theory by asking whether increasing acetylcholine levels during early sleep would disrupt motor memory consolidation. We trained separate groups of adult mice on the rotarod walking and skilled reaching for food tasks, and after training, administered physostigmine, an acetylcholinesterase inhibitor, to increase cholinergic tone in subsequent sleep. Post-sleep testing suggested that physostigmine impaired motor skill acquisition. Home-cage video monitoring and electrophysiology revealed that physostigmine disrupted sleep structure, delayed non-rapid-eye-movement sleep onset, and reduced slow-wave power in the hippocampus and cortex. The impaired motor performance with physostigmine, however, was not solely due to its effects on sleep structure, as one hour of sleep deprivation after training did not impair rotarod performance. A reduction in cholinergic tone by inactivation of cholinergic neurons during early sleep also affected rotarod performance. Administration of agonists and antagonists of muscarinic and nicotinic acetylcholine receptors revealed that activation of muscarinic receptors during early sleep impaired rotarod performance. The experiments suggest that the increased slow wave activity and inactivation of muscarinic receptors during early sleep due to reduced acetylcholine contribute to motor memory consolidation.