دکتر محمد جوان

Demyelination and inflammation are the most common pathobiological manifestations contributing to depression in multiple sclerosis (MS).

The current study aimed to evaluate the effects of acetyl-L-carnitine (ALC) in attenuating depressive-like symptoms in the cuprizone intoxication mouse model.

C57BL/6 mice were categorized into three groups (n = 6 each) as follows: The control animals (CTL), the cuprizone-intoxicated mice (CPZ), and the group that received cuprizone and acetyl-L-carnitine (CPZ+ALC). Depressive-like behaviors were evaluated by the forced swim test (FST) and the tail suspension test (TST). The prefrontal cortex (PFC) and corpus callosum (CC) areas were assessed in terms of histopathology, biochemistry, and gene expression.

Following oral gavage of ALC, the immobility time, which represents the despairing time, significantly decreased compared to the CPZ group. Histopathological evaluation showed that remyelination in the CC increased significantly in animals receiving ALC compared to the CPZ mice. Acetyl-L-carnitine considerably decreased the nitric oxide (NO) level in the PFC of the brain in the demyelinated mice compared to the CPZ group. The qRT-PCR results revealed that ALC significantly increased neuronal nitric oxide synthase (nNOS) expression but decreased inducible nitric oxide synthase (iNOS) gene expression compared to the CPZ group.

Acetyl-L-carnitine attenuated depressive-like behaviors in the cuprizone demyelination mouse model of MS. These neuroprotective effects of ALC may be exerted by facilitating the remyelination process and modulating the NO level in the PFC.

Aging of can lead to significant cognitive and neurobehavioral deficits. In addition, aging leads to more susceptibility to neurological disorders, such as stroke, traumatic brain injury, and neurodegeneration. Accordingly, white matter (WM) changes associated with aging may be a factor in the functional impairment seen in the elderly. In this study, we initially determined whether the corpus callosum (CC) of old mice exhibited signs of cellular aging compared to young mice. To investigate cellular aging indices we examined SA-β-galactosidase and relative telomere length as markers of aging in the CC. Following this, we measured the myelination index through the g-ratio calculation. Our study demonstrated an increased g-ratio and axon diameter in aged mice. We also analyzed ultrastructural changes of myelinated axons and mitochondria in the CC of aged mice. The CC underwent substantial ultrastructural variation following the aging. These changes included myelination breakdown, the formation of myelin balloons, loss of the compact structure of myelin, and increased intramembrane density. we also investigated the impact of aging on mitochondria ultrastructure. We observed the presence of dark matrices and interconnected crista in a subgroup of the mitochondria in the CC. Such alterations are indicative of the deterioration in the integrity of WM with age. These findings are crucial as they provide insights into how aging affects the structural and functional aspects of WM, particularly in the CC. Understanding these changes is essential for developing strategies to mitigate age-related cognitive decline and to address the heightened susceptibility of aged WM to neurological disorders.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. The autoimmune pathology and long-term inflammation lead to substantial demyelination. These events lead to a substantial loss of oligodendrocytes (OLs), which in a longer period, results in axonal loss and long-term disabilities. Neural cells protection approaches decelerate or inhibit the disease progress to avoid further disability. Previous studies showed that metformin has beneficial effects against neurodegenerative conditions. In this study, we examined possible protective effects of metformin on toxin-induced myelin destruction in adult mice brains.

In this experimental study, lysophosphatidylcholine (LPC) was used to induce demyelination in mice optic chiasm. We examined the extent of demyelination at different time points post LPC injection using myelin staining and evaluated the severity of inflammation. Functional state of optic pathway was evaluated by visual evoked potential (VEP) recording.

Metformin attenuated LPC-induced demyelination (P<0.05) and inflammation (P<0.05) and protected against significant decrease (P<0.05) in functional conductivity of optic tract. These data indicated that metformin administration attenuates the myelin degeneration following LPC injection which led to functional enhancement.

Our findings suggest metformin for combination therapy for patients suffering from the myelin degenerative diseases, especially multiple sclerosis; however, additional mechanistic studies are required.

The modulatory effect of deep inspiration (DI) on airway constriction is impaired in asthma. However, mechanisms underlying this impairment are not clear. Since there is evidence indicating that Rho-kinase activation mediates force maintenance under oscillatory strain, we investigated the impact of Rho-kinase inhibition on the bronchodilatory effect of DI in ovalbumin (OVA) sensitized guinea pigs.

forty-eight male Dunkin Hartley guinea pigs were divided into 8 groups including saline/ constant, saline/DI, OVA/constant, OVA/DI, Rho-I/OVA/constant, Rho-I/OVA/DI, OVA-Rho-I/MCh/constant, and OVA-Rho-I/MCh/DI. Animals were subjected to 12 inhalations of OVA or saline aerosol. Guinea pigs in Rho-I/OVA/constant or DI groups were treated with the Rho-kinase inhibitor (Rho-I) (Y-27632, 1 mM aerosols) prior to the last 8 allergen inhalations and OVA-Rho-I/MCh/constant or DI groups received Y-27632 at the end of allergen sensitization protocol before methacholine challenge. The bronchodilatory effect of DI in guinea pigs that were exposed to methacholine was assessed by using an animal ventilator. The bronchodilatory effect was assessed using several parameters: the airway pressure maintenance, airway pressure recovery, and decline of airway pressure.

Results indicated that application of Y-27632 prior to methacholine challenge reduces the airway smooth muscle ability to maintain pressure and also causes further decline in airway pressure in OVA-sensitized animals undergone DI. However, the inhibition of Rho-kinase before OVA inhalations had minimal effect.

We propose that alteration of Rho-kinase signaling pathway may be one of the mechanisms underlying the impairment of DI-induced bronchodilation in OVA-sensitized guinea pigs.

Spinal cord injury (SCI) is a condition which can lead to permanent loss of neurons, glial and precursor cells. According to the positive influences of electrical stimulation in the neurogenesis, we hypothesized that sub-threshold electrical stimulation in the presence of exogenous astrocyte may trigger the differential regulation of wingless-type3 (Wnt-3) and eukaryotic initiation factor-2α (eIF2α) mediators in spinal cord injured rats.

Forty male Wistar rats (weighing 250-280g) were randomly divided into four groups: sham, SCI, SCI+astrocyte and SCI+astrocyte which followed by electrical stimulation. We evaluated the glial fibrillary acidic protein (GFAP), doublecortin, Wnt-3 and eIF2α proteins by immunofluorescence and immunoblotting techniques.

The results show that expression of Wnt-3 and eIF2α proteins significantly enhanced after 14 days in the electrical stimulation+ SCI+astrocyte group in comparison with SCI and SCI+astrocyte groups. Also, the expression of GFAP cells was significantly increased after 14 days by electrical stimulation compared with other groups. Electrical stimulation had no effect on expression of doublecortin after 14 days.

This survey demonstrates that sub-threshold electrical stimulation up-regulates Wnt-3 and eIF2α mediators. Also, GFAP marker expression has been increased in animals subjected to electrical stimulation. But there are no evidences based on doublecortin expression as a neurogenesis biomarker.

Neuronal survival in multiple sclerosis (MS) and other demyelinating diseases depends on the preservation of myelin and remyelination of axons. Myelin protection is the main purpose to decrease myelin damage in the central nervous system (CNS). Ursolic acid (UA) as a natural product in apple is suggested to protect neural cells. This study is the first to demonstrate an effect for UA on CNS myelin loss induced by cuprizone toxin. In the current study, we hypothesized that daily treatment with UA in drinking water (1 mg/ml) prevents myelin damage by 6 weeks administration of CPZ in mice pellet which lead to corpus callosum axonal demyelination. We assessed the myelin content and the number of myelinating cells in corpus callosum by FluoroMyelin and luxol fast blue staining as well as by immunostaining against MBP and Olig2. Our finding indicated that UA could decrease the extent of demyelination area and enhanced myelin stain intensity within CC and protected oligodendrocyte lineage cells against cuprizone toxin. We could conclude that myelinated structures could be protected by UA in corpus callosum, which provide favorable evidence for the possibility of application of UA in demyelinating diseases and traumatic injuries.

Synaptic plasticity has been suggested as the primary physiological mechanism underlying memory formation. Many experimental approaches have been used to investigate whether the mechanisms underlying Long-Term Potentiation (LTP) are activated during learning. Nevertheless, little evidence states that hippocampal-dependent learning triggers synaptic plasticity. In this study, we investigated if learning and memory in the Barnes maze test are accompanied by the occurrence of LTP in Schaffer collateral to CA1 synapses in freely moving rats.

The rats were implanted with a recording electrode in stratum radiatum and stimulating electrodes in Schaffer collaterals of the CA1 region in the dorsal hippocampus of the right hemisphere. Following the recovery period of at least 10 days, field potentials were recorded in freely moving animals before and after training them in Barnes maze as a hippocampal-dependent spatial learning and memory test. The slope of extracellular field Excitatory Postsynaptic Potentials (fEPSPs) was measured before and after the Barnes maze test.

The results showed that the fEPSP slope did not change after learning and memory in the Barnes maze test, and this spatial learning did not result in a change in synaptic potentiation in the CA1 region of the hippocampus.

Spatial learning and memory in the Barnes maze test are not accompanied by LTP induction in Schaffer collateral-CA1 synapses.

The generation of oligodendrocyte progenitor cells (OPCs) offers tremendous opportunities for cell replacement therapy in demyelinating diseases such as multiple sclerosis (MS) and spinal cord injury. Recently, the prospect of reprogramming terminally differentiated adult cells towards another mature somatic cell or progenitor cells without an intermediate pluripotent state has been of interest. Trichostatin A is a histone deacetylase inhibitor which opens the chromatin and facilitates the transcription of silence genes. In this study, we have treated human astrocytes line U87 and primary culture of mouse astrocytes with TSA for 12 hours, prior their transfer to OPC induction medium. Then we evaluated the morphology and the fate of the treated astrocytes at post-treatment days. Both cell lines acquired OPC morphology and expressed OPC specific markers. Following transfer to differentiation medium, U87-derived iOPCs differentiated to oligodendrocyte like cells and expressed PLP as a mature oligodendrocyte marker. Our results introduce TSA as an inducer for production of OPCs from astrocytes and can be considered a potential way for the treatment of demyelinating diseases.

Multiple sclerosis (MS) is an autoimmune disease which affects myelin in the central nervous system (CNS) and leads to serious disability. Currently available treatments for MS mainly suppress the immune system. Regenerative medicine-based approaches attempt to increase myelin repair by targeting endogenous progenitors or transplanting stem cells or their derivatives. Fingolimod exerts anti-inflammatory effects and directly affects neural cells. In this study we assessed the effect of fingolimod on transplanted human induced pluripotent stem cell derived neural progenitors (hiPSC-NPs). hiPSC-NPs were labeled by green fluorescence protein (GFP) and transplanted into the corpus callosum of mice which were chronically demyelinated after cuprizone (CPZ) feedings for 10 weeks. The animals received fingolimod from 1 day prior to NPs transplantation via gavage as well as daily intraperitoneal cyclosporine A from 2 days before cell transplantation until the time of sampling. At either 7 or 21 days after NPs transplantation, the animals were sacrificed and their brains were histologically evaluated for the number of transplanted cells and their fate. In the animals treated with fingolimod, we observed higher numbers of NPs within the injection site compared to the animals who did not receive fingolimod showing that hiPSC- NPs were more efficiently differentiated to the oligodendrocyte lineage. These data have suggested that repetitive treatment with fingolimod, beside its anti-inflammatory effect, may enhance the survival and differentiation of transplanted NPs to oligodendrocyte lineage cells to participate in myelin repair.

Nogo-A and Nogo receptor (NgR) are expressed in the subventricular zone (SVZ) stem cells. NgR plays critical inhibitory roles in axonal regeneration and remyelination. However, the role of NgR in SVZ niche behaviors in demyelination context is still uncertain. Here we investigated the effects of NgR inhibition on SVZ niche reaction in a local model of demyelination in adult mouse optic chiasm.
Demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. We injected siRNAs against NgR intracerebroventricularly via a permanent cannula over 14 days to knockdown NgR. To trace SVZ stem cells and assess the effect of NgR inhibition on their reaction, BrdU was injected to the animals prior to the demyelination induction. Immunohistochemistry and histological analysis was carried out 3, 7 and 14 days post demyelination lesion.
NgR inhibition significantly increased the numbers of proliferating cells in SVZ in response to demeylination. The number of BrdUﳩ抃澩꺈ẞ⭨ cells in the neurogenic zone of the lateral ventricles was enhanced when NgR was blocked. These progenitor cells (Olig2, GFAP or PSA-NCAM) were mobilized away from this SVZ as a function of time. Inhibition of NgR significantly reduced demyelination extension in optic chiasm.
Our findings reveal that inhibition of NgR potentiates adult SVZ progenitor cells proliferation and differentiation in demyelination condition and facilitates remyelination in the optic chiasm. Therefore, inhibition of NgR function could have therapeutic potential for demyelinating disease like multiple sclerosis.

Due to recent progress in production of human embryonic stem cell-derived oligodendrocyte progenitor cells (hESC-OPCs) for ameliorating myelin disease such as multiple sclerosis (MS) and the role of purinergic signaling in OPCs development, we avaluated the profile of purinergic receptors expression during development of OPCs from hESC.
In this experimental study, we used reverse transcription and quantitative polymerase chain reaction (RT-qPCR) to obtain more information about potential roles of purinergic receptors during in vitro production of hESC-OPCs. We first determined the expression level of different subtypes of purinergic receptors in hESCs, embryoid bodies (EBs), and hESC-OPCs. The effects of A1adenosine receptor (A1AR) activation on hESC-OPCs development were subsequently examined.
hESCs and OPCs had different mRNA expression levels of the AR subtypes. ARs mRNA were expressed in the EB stage, except for A2AAR. We observed expressions of several P2X (P2X1, 2, 3, 4, 5, 7) and P2Y (P2Y1, 2, 4, 6, 11-14) genes in hESCs. hESC-OPCs expressed different subtypes of P2X (P2X1, 2, 3,4,5,7) and P2Y (P2Y1, 2, 4, 6, 11-14). Except for P2X1 and P2X6, all other P2X and P2Y purinergic receptor subtypes expressed in EBs. We also indicate that A1AR might be involved in modulating gene expression levels of cell cycle regulators in an agonist and/or dose-dependent manner.
Elucidation of the expression pattern of purinergic receptors and the effects of different subtypes of these receptors in hESC-OPCs may have a promising role in future cell-based therapy or drug design for demyelinating disease.

Recent studies have shown that intermittent normobaric hyperoxia (HO) protects the rat brain from ischemia reperfusion injury. However, the exact mechanism of this kind of protection in vivo is not known. In this study, the effect of HO on expression of TNFR1 and TNFR2 in a stroke model was investigated.
In this experimental study, rats were divided into 4 groups: normoxia – sham, hyperoxia – sham, normoxia – stroke and hyperoxia –stroke for each factor (TNFR1 or TNFR2). Hyperoxia groups were exposed to 95% inspired oxygen for 4 h/day and 6 consecutive days. Oxygen concentration in the control groups was 21% (normoxia, room air). After 24h, the rats were subjected to 60 min of right middle cerebral artery occlusion (MCAO). After 24h reperfusion, neurological deficit scores (NDS) and TNFR1, 2 brain levels using Western Blot were assessed.
Preconditioning with HO decreased NDS. Also, followed by stroke and reperfusion, TNFR1 levels significantly increased; while there was no significant difference in hyperoxia groups compared with normoxia groups in the cortex, HO significantly reduced TNFR1 expression in subcortex. On the other hand, groups of stroke compared to sham groups significantly expressed lower levels of TNFR2 in the cortex and subcortex. There was no significant difference in hyperoxia groups compared with normoxia groups in these areas.
Although additional studies will be required to further elucidate precise mechanisms of ischemic tolerance, it seems that HO is associated with the expression of TNFR1 in subcortex, consistent with an active role in the genesis of ischemic protection.

Spinal cord injuries are accompanied with significant demyelination of axons and subsequent locomotor dysfunction. To identify the extent of damage following electrolytic lesion of ventrolateral white matter, essential area for initiation of locomotor activity, we assessed demyelination as well as alteration in motor performance. Moreover, the protective effect of estradiol as a candidate treatment for preservation of myelin and locomotor activity after injury was examined due to its anti-apoptotic and anti-inflammatory activities.
A unilateral electrolytic lesion positioned in the right ventrolateral funiculus (VLF) was applied following laminectomy at T8-T9. In the estradiol-treated injury group, animals received a pharmacological single dose of estradiol valerate (4 mg/kg) at 30min post injury. Locomotor function was assessed using rotarod and open field tasks during 4 weeks after injury.
Obtained results showed significant demyelination at the site of injury and caudal areas following lesion as well as altered motor performance. Post-spinal cord injury administration of estradiol enhanced white matter maintenance at the site of lesion, restored the level of myelin basic protein (MBP), decreased TUNEL positive cells and improved functional recovery.
Taken together, these results indicate that demyelination after lesion in VLF may be a contributing factor to limited motor performance, and suggest that pharmacological doses of estradiol may have an early protective effect through sparing of white matter.

Memory and cognitive impairments are some of devastating outcomes of Multiple Sclerosis (MS) plaques in hippocampus, the gray matter part of the brain. The present study aimed to evaluate the intrahippocampal injection of Ethidium Bromide (EB) as a simple and focal model to assess cognition and gray matter demyelination. Thirty Wistar rats were divided into three groups: control group, which received saline, as solvent of EB, into the hippocampus; and two experimental groups, which received 3 μL of EB into the hippocampus, and then, were evaluated 7 and 28 days after EB injection (n=10 in each group), using a 5-day protocol of Morris Water Maze (MWM) task as well as Transmission Electron Microscopy (TEM) assay. Seven days after EB injection, the behavioral study revealed a significance increase in travelled distance for platform finding in the experimental group compared to the control group. In addition, the nucleus of oligodendrocyte showed the typical clumped chromatin, probably attributed to apoptosis, and the myelin sheaths of some axons were unwrapped and disintegrated. Twentyeight days after EB injection, the traveled distance and the time spent in target quadrant significantly decreased and increased, respectively in experimental groups compared to the control group. Also, TEM micrographs revealed a thin layer of remyelination around the axons in 28 days lesion group. While intracerebral or intraventricular injection of EB is disseminated in different parts of the brain and can affect the other motor and sensory systems, this model is confined locally and facilitates behavioral study. Also, this project could show improvement of memory function subsequent to the physiological repair of the gray matter of the hippocampus.

Hippocampal insults have been observed in multiple sclerosis (MS) patients. Fibroblast growth factor-2 (FGF2) induces neurogenesis in the hippocampus and enhances the proliferation, migration and differentiation of oligodendrocyte progenitor cells (OPCs). In the current study, we have investigated the effect of FGF2 on the processes of gliotoxin induced demyelination and subsequent remyelination in the hippocampus. In this experimental study adult male Sprague-Dawley rats received either saline or lysolecithin (LPC) injections to the right hippocampi. Animals received intraperitoneal (i.p.) injections of FGF2 (5 ng/g) on days 0, 5, 12 and 26 post-LPC. Expressions of myelin basic protein (Mbp) as a marker of myelination, Olig2 as a marker of OPC proliferation, Nestin as a marker of neural progenitor cells, and glial fibrillary acidic protein (Gfap) as a marker of reactive astrocytes were investigated in the right hippocampi by reverse transcriptase-polymerase chain reaction (RT-PCR). There was reduced Mbp expression at seven days after LPC injection, increased expressions of Olig2 and Nestin, and the level of Gfap did not change. FGF2 treatment reversed the expression level of Mbp to the control, significantly enhanced the levels of Olig2 and Nestin, but did not change the level of Gfap. At day-28 post- LPC, the expression level of Mbp was higher than the control in LPC-treated animals that received FGF2. The levels of Olig2, Nestin and Gfap were at the control level in the non-treated LPC group but significantly higher in the FGF2-t reated LPC group. FGF2 enhanced hippocampal myelination and potentiated the recruitment of OPCs and neural stem cells (NSCs) to the lesion area. Long-term application of FGF2 might also enhance astrogliosis in the lesion site.

In addition to its antioxidant effect, Vitamin E or α–tocopherol is suggested to enhance remyelination in the animal model of non-inflammatory demyelination. In this study, the possible proliferative effect of vitamin E on human- induced pluripotent stem cell-derived neural progenitors (hiPS-NPs) and the underlying mechanisms were investigated in vitro. NPs were induced from iPS cells via 3 steps within 18 days and then characterized for NPs markers NESTIN, SOX1 and OTX2. MTT assay was used to compare cell populations. LY294002, U0126 and PP2 were used for selective inhibition of enzymes PI3K, MEK and Src-kinase, respectively. Vitamin E increased hiPS-NPs proliferation after 24 and 48 h exposure. The inhibition of both PI3K/Akt and Src-kinase signaling pathways counteracted the effect of vitamin E of NPs. Our data suggest that vitamin E may enhance NPs proliferation via activating PI3K and Src-kinase and may enhance myelin repair following demyelinating Injuries.

Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid (VPA) are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction (PCR) was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell (NSC) markers. Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog (p<0.05). Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc (p<0.001). VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc (p<0.01), Pax6 and Sox1 (p<0.001). These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4.