Olalekan Michael Ogundele

Fluoride and aluminium disturb neuronal morphology, transport functions, cholinesterase, lysosomal and cell cycle activities

UNLABELLED Fluoride and aluminium have been reported to cause severe alterations in the brain. However, their exact mechanisms of neurotoxic activities remain unknown. AIM This study was designed to investigate the role of fluoride and aluminium in neuronal transport, lysosomal, cell cycle protein and acetylcholinesterase activities. METHOD Adult Wistar rats were given low and high doses of fluoride, aluminium and a combination of both with the control group receiving distilled water for 30 days. Blood sera and brain homogenates were quantified for alkaline phosphatase (biomarker for neuronal transport) activities. Brain sections were stained with cresyl fast violet to detect neuronal cell damage. Histochemical demonstration of acetylcholinesterase (AChE) activity and the immunohistochemical detection of cell cycle protein (anti-cyclin D) and lysosomal protein (anti-cathepsin D) were done using the antigen retrieval method. RESULT Results showed severe histomorphologic alterations, dysregulation of membrane transport activities, inhibition of AChE activities and increased expression of lysosomal and cell cycle proteins. CONCLUSION These findings confirm that excessive fluoride and aluminium intake induces the progression of cell death which inhibit AChE activities and trigger the release of lysosomal and cell cycle proteins.

Interplay of glia activation and oxidative stress formation in fluoride and aluminium exposure

BACKGROUND Oxidative stress formation is pivotal in the action of environmental agents which trigger the activation of glial cells and neuroinflammation to stimulate compensatory mechanisms aimed at restoring homeostasis. AIM This study sets to demonstrate the interplay of fluoride (F) and aluminium (Al) in brain metabolism. Specifically, it reveals how oxidative stress impacts the activation of astrocytes (GFAP), mediates proinflammatory responses (microglia and B-cells: CD68 and CD 20 respectively) and shows the pattern of lipid peroxidation in the brain following fluoride and (or) aluminium treatment in vivo. METHOD Male adult Wistar rats were treated with low and high doses of fluoride, aluminium or combination of fluoride-aluminium for 30 days. The control group received distilled water for the duration of the treatment. Blood and brain tissue homogenates were prepared for colorimetric assay of stress biomarkers [malonialdehyde (MDA) and superoxide dismutase (SOD)]. Subsequent analysis involved immunodetection of astrocytes (anti-GFAP), microglial (anti-CD68) and B-cells (anti-CD20) in coronal sections of the prefrontal cortex using antigen retrieval immunohistochemistry. RESULT AND CONCLUSION Aluminium, fluoride and a combination of aluminium-fluoride treatments caused an increase in brain lipid peroxidation products and reactive oxygen species (ROS) formation. Similarly, an increase in glial activation and inflammatory response were seen in these groups versus the control. Oxidative stress induced glial activation (GFAP) and increased the expression of B cells (CD20). This also corresponded to the extent of tissue damage and lipid peroxidation observed. Taken together, the results suggest a close link between oxidative stress neuroinflamation and degeneration in aluminium-fluoride toxicity.

−NMDA R/+VDR pharmacological phenotype as a novel therapeutic target in relieving motor–cognitive impairments in Parkinsonism

Abstract Background: Parkinsonism describes Parkinson’s disease and other associated degenerative changes in the brain resulting in movement disorders. The motor cortex, extrapyramidal tracts and nigrostriatal tract are brain regions forming part of the motor neural system and are primary targets for drug or chemotoxins induced Parkinsonism. The cause of Parkinsonism has been described as wide and elusive, however, environmental toxins and drugs accounts for large percentage of spontaneous cases in humans. A common mechanism in the cause and progression of drug/chemotoxin induced Parkinsonism involves calcium signalling in; oxidative stress, autophagy, cytoskeletal instability and excitotoxicity .Aim: This study sets to investigate the effect of targeting calcium controlling receptors, specifically activation of Vitamin D3 receptor (VDR) and inhibition of N-Methyl-D-Aspartate Receptor (NMDAR) in the motor cortex of mice model of drug induced Parkinsonism. Also we demonstrated how these interventions improved neural activity, cytoskeleton, glia/neuron count and motor–cognitive functions in vivo. Methods: Adult mice were separated into six groups of n = 5 animals each. Body weight (5 mg/kg) of haloperidol was administered intraperitoneally for 7 days to block dopaminergic D2 receptors and induce degeneration in the motor cortex following which an intervention of VDR agonist (VDRA), and (or) NMDAR inhibitor was administered for 7 days. A set of control animals received normal saline while a separate group of control animals received the combined intervention of VDRA and NMDAR inhibitor without prior treatment with haloperidol. Behavioral tests for motor and cognitive functions were carried out at the end of the treatment and intervention periods. Subsequently, neural activity in the motor cortex was recorded in vivo using unilateral wire electrodes. We also employed immunohistochemistry to demonstrate neuron, glia, neurofilament and proliferation in the motor cortex after haloperidol treatment and the intervention. Result/Discussion: We observed a decline in motor function and memory index in the haloperidol treatment group when compared with the control. Similarly, there was a decline in neural activity in the motor cortex (a reduced depolarization peak frequency). General cell loss (neuron and glia) and depletion of neurofilament were characteristic anatomical changes seen in the motor cortex of this group. However, Vitamin D3 intervention facilitated an improvement in motor–cognitive function, neural activity, glia/neuron survival and neurofilament expression. NMDAR inhibition and the combined intervention improved motor–cognitive functions but not as significant as values observed in VDRA intervention. Interestingly, animals treated with the combined intervention without prior haloperidol treatment showed a decline in motor function and neural activity. Conclusion: Our findings suggest that calcium mediated toxicity is primary to the cause and progression of Parkinsonism and targeting receptors that primarily modulates calcium reduces the morphological and behavioral deficits in drug induced Parkinsonism. VDR activation was more effective than NMDAR inhibition and a combined intervention. We conclude that targeting VDR is key for controlling calcium toxicity in drug/chemotoxin induced Parkinsonism.

Cerebrovascular changes in the rat brain in two models of ischemia

BACKGROUND Vascular occlusion and cyanide neurotoxicity induces oxidative stress and degeneration in the brain. This oxidant induced stress changes the vascular dynamics of cerebral blood vessels, and participates in homeostatic response mechanisms which balance oxygen supply to hypoxic stress-sensitive neurons. The associated changes in vascular morphology include remodeling of the microvasculature and endothelial changes, alterations in regional circulation and variations in the blood brain barrier (BBB). This study compares alterations in physiology of the cerebral artery after a short-term oxidative stress induced by cyanide toxicity and vascular occlusion. METHOD Adult Wistar rats (N=30) were divided into three groups; vascular occlusion (VO) (n=12), potassium cyanide administration (CN) (n=12) and Control-CO (n=6). The CN rates were treated with 30mg/kg of orally administered KCN while the VO was subjected to global vascular occlusion, both for a duration of 10 days, described as the treatment phase. Control animals were fed on normal rat chow and water for 10 days. At the end of the treatment phase, n=6 animals in each of the VO, CN and VO groups were anesthetized with sodium pentobarbital (50IP) and the CCA exposed, after which pin electrodes were implanted to record the spikes form the tunica media of the CCA. After day 10, treatment was discontinued for these animals, each remaining in the VO and CN groups (VO-I and CN-I) until day 20 (withdrawal phase) following which the spikes were recorded using the procedure described above. RESULTS/DISCUSSION Vascular occlusion and cyanide toxicity increased vascular resistance in the MCA (reduced lumen thickness ratio) and increased the diameter of the CCA after the treatment phase of 10 days. After 10 days of withdrawal, the VO group showed a reduction in resistance and an increase in the lumen width/wall thickness ratio (LWR) while the CN group showed increased resistance and a reduction in LWR. CONCLUSION Cyanide toxicity increased vascular resistance by inducing degenerative changes in the wall of the artery while vascular occlusion increased resistance through mechanical stress and increased thickness of arterial wall. After the withdrawal phase, vascular resistance diminished in the VO to a significantly greater extent than the CN.

IGF-I Gene Therapy in Aging Rats Modulates Hippocampal Genes Relevant to Memory Function

In rats, learning and memory performance decline during normal aging, which makes this rodent species a suitable model to evaluate therapeutic strategies. In aging rats, insulin-like growth factor-I (IGF-I), is known to significantly improve spatial memory accuracy as compared to control counterparts. A constellation of gene expression changes underlie the hippocampal phenotype of aging but no studies on the effects of IGF-I on the hippocampal transcriptome of old rodents have been documented. Here, we assessed the effects of IGF-I gene therapy on spatial memory performance in old female rats and compared them with changes in the hippocampal transcriptome. In the Barnes maze test, experimental rats showed a significantly higher exploratory frequency of the goal hole than controls. Hippocampal RNA-sequencing showed that 219 genes are differentially expressed in 28-month-old rats intracerebroventricularly injected with an adenovector expressing rat IGF-I as compared with placebo adenovector-injected counterparts. From the differentially expressed genes, 81 were down and 138 upregulated. From those genes, a list of functionally relevant genes, concerning hippocampal IGF-I expression, synaptic plasticity as well as neuronal function was identified. Our results provide an initial glimpse at the molecular mechanisms underlying the neuroprotective actions of IGF-I in the aging brain.

Long-lasting training in the Barnes maze prompts hippocampal spinogenesis and habituation in rats.

There is a constant need to assess spatial memory in small rodents to elucidate the basics of cognition in neuroscience experiments. Thus, the significance of the Barnes maze in the biology of hippocampal and cortical neural function cannot be overemphasized. Despite the wide use of the Barnes maze, the effect of maze task training on the structure of hippocampal neurons is yet to be elucidated. Adult Sprague-Dawley rats were subjected to intense training on the Barnes maze (3 months). Subsequently, the hippocampus (cornus ammonis and dentate gyrus) of separate sets of rats was processed for Golgi Colonnier techniques (silver impregnation) and adenoviral-green fluorescent protein labeling (immunohistochemistry). Our results showed that training the animals on the Barne maze increased spinogenesis significantly in the cornus ammonis and dentate gyrus neurons. In addition, we identified a critical time point at which the rats habituated to the trial without escaping box (the probe trial) and could not be tested further in the maze. Taken together, we deduced that a prolonged test on the dry land maze facilitated habituation and caused an increase in hippocampal dendritic spine count. As such, the dry land maze is a suitable paradigm for assessing spatial memory in rats. However, precautions should be taken in selecting suitable experimental controls on the basis of the duration of a study.

Motor and memory function in rat models of cyanide toxicity and vascular occlusion induced ischemic injury

Although oxidative stress is characteristic of global vascular occlusion and cyanide toxicity, the pattern of cerebral metabolism reconditioning and rate of progression or reversal of neural tissue damage differ for both forms of ischemia. Thus, it is important to compare cognitive and motor functions in both models of ischemia involving cyanide treatment (CN) and vascular occlusion (VO). Adult Wistar rats (N=30) were divided into three groups; VO (n=12), CN (n=12) and Control-CO (n=6). The CN was treated with 30mg/Kg of potassium cyanide (KCN); VO was subjected to global vascular occlusion-both for duration of 10 days. The control (CO) was fed on normal rat chow and water for the same duration. At day 10, the test and control groups (CN, VO and CO) were subjected to motor function tests (Table edge tests and Open Field Test) and memory function tests (Y-Maze and Novel object recognition) while the withdrawal groups CN-I and VO-I were subjected to the same set of tests at day 20 (the withdrawal phase). The results show that both cyanide toxicity and vascular occlusion caused a decline in motor and memory function when compared with the control. Also, the cyanide treatment produced a more rapid decline in these behavioral parameters when compared with the vascular occlusion during the treatment phase. After the withdrawal phase, cyanide treatment (CN-I) showed either an improvement or restoration of motor and memory function when compared to the CN and control. Withdrawal of vascular occlusion caused no improvement, and in some cases a decline in motor and memory function. In conclusion, cyanide toxicity caused a decline in motor and memory function after the treatment while vascular occlusion caused no significant decline in cognition and motor function at this time. After the withdrawal phase, the effect of cyanide toxicity was reduced and significant improvements were observed in the behavioral tests (motor and cognitive), while a decline in these functions were seen in the vascular occlusion group after this phase.

CaMKIIα expression in a mouse model of NMDAR hypofunction schizophrenia: Putative roles for IGF-1R and TLR4

Schizophrenia (SCZ) is a neuropsychiatric disorder that is linked to social behavioral deficits and other negative symptoms associated with hippocampal synaptic dysfunction. Synaptic mechanism of schizophrenia is characterized by loss of hippocampal N-Methyl-d-Aspartate Receptor (NMDAR) activity (NMDAR hypofunction) and dendritic spines. Previous studies show that genetic deletion of hippocampal synaptic regulatory calcium-calmodulin dependent kinase II alpha (CaMKIIα) cause synaptic and behavioral defects associated with schizophrenia in mice. Although CaMKIIα is involved in modulation of NMDAR activity, it is equally linked to inflammatory and neurotropin signaling in neurons. Based on these propositions, we speculate that non-neurotransmitter upstream receptors associated with neurotropic and inflammatory signaling activities of CaMKIIα may alter its synaptic function. Besides, how these receptors (i.e. inflammatory and neurotropic receptors) alter CaMKIIα function (phosphorylation) relative to hippocampal NMDAR activity in schizophrenia is poorly understood. Here, we examined the relationship between toll-like receptor (TLR4; inflammatory), insulin-like growth factor receptor 1 (IGF-1R; neurotropic) and CaMKIIα expression in the hippocampus of behaviorally deficient schizophrenic mice after we induced schizophrenia through NMDAR inhibition. Schizophrenia was induced in WT (C57BL/6) mice through intraperitoneal administration of 30mg/Kg ketamine (NMDAR antagonist) for 5days (WT/SCZ). Five days after the last ketamine treatment, wild type schizophrenic mice show deficiencies in sociability and social novelty behavior. Furthermore, there was a significant decrease in hippocampal CaMKIIα (p<0.001) and IGF-1R (p<0.001) expression when assessed through immunoblotting and confocal immunofluorescence microscopy. Additionally, WT schizophrenic mice show an increased percentage of phosphorylated CaMKIIα in addition to upregulated TLR4 signaling (TLR4, NF-κB, and MAPK/ErK) in the hippocampus. To ascertain the functional link between TLR4, IGF-1R and CaMKIIα relative to NMDAR hypofunction in schizophrenia, we created hippocampal-specific TLR4 knockdown mouse using AAV-driven Cre-lox technique (TLR4 KD). Subsequently, we inhibited NMDAR function in TLR4 KD mice in an attempt to induce schizophrenia (TLR4 KD SCZ). Interestingly, IGF-1R and CaMKIIα expressions were preserved in the TLR4 KD hippocampus after attenuation of NMDAR function. Furthermore, TLR4 KD SCZ mice showed no prominent defects in sociability and social novelty behavior when compared with the control (WT). Our results show that a sustained IGF-1R expression may preserve the synaptic activity of CaMKIIα while TLR4 signaling ablates hippocampal CaMKIIα expression in NMDAR hypofunction schizophrenia. Together, we infer that IGF-1R depletion and increased TLR4 signaling are non-neurotransmitter pro-schizophrenic cues that can reduce synaptic CaMKIIα activity in a pharmacologic mouse model of schizophrenia.

Systemic Sympathoexcitation Was Associated with Paraventricular Hypothalamic Phosphorylation of Synaptic CaMKIIα and MAPK/ErK

Systemic administration of adrenergic agonist (Isoproterenol; ISOP) is known to facilitate cardiovascular changes associated with heart failure through an upregulation of cardiac toll-like receptor 4 (TLR4). Furthermore, previous studies have shown that cardiac tissue-specific deletion of TLR4 protects the heart against such damage. Since the autonomic regulation of systemic cardiovascular function originates from pre-autonomic sympathetic centers in the brain, it is unclear how a systemically driven sympathetic change may affect the pre-autonomic paraventricular hypothalamic nuclei (PVN) TLR4 expression. Here, we examined how change in PVN TLR4 was associated with alterations in the neurochemical cytoarchitecture of the PVN in systemic adrenergic activation. After 48 h of intraperitoneal 150 mg/kg ISOP treatment, there was a change in PVN CaMKIIα and MAPK/ErK expression, and an increase in TLR4 in expression. This was seen as an increase in p-MAPK/ErK, and a decrease in synaptic CaMKIIα expression in the PVN (p < 0.01) of ISOP treated mice. Furthermore, there was an upregulation of vesicular glutamate transporter (VGLUT 2; p < 0.01) and a decreased expression of GABA in the PVN of Isoproterenol (ISOP) treated WT mice (p < 0.01). However, after a PVN-specific knockdown of TLR4, the effect of systemic administration of ISOP was attenuated, as indicated by a decrease in p-MAPK/ErK (p < 0.01) and upregulation of CaMKIIα (p < 0.05). Additionally, loss of inhibitory function was averted while VGLUT2 expression decreased when compared with the ISOP treated wild type mice and the control. Taken together, the outcome of this study showed that systemic adrenergic activation may alter the expression, and phosphorylation of preautonomic MAPK/ErK and CaMKIIα downstream of TLR4. As such, by outlining the roles of these kinases in synaptic function, we have identified the significance of neural TLR4 in the progression, and attenuation of synaptic changes in the pre-autonomic sympathetic centers.

Thalamic dopaminergic neurons projects to the paraventricular nucleus-rostral ventrolateral medulla/C1 neural circuit

Paraventricular nuclei (PVN) projections to the rostral ventrolateral medulla (RVLM)/C1 catecholaminergic neuron group constitute the pre‐autonomic sympathetic center involved in the neural control of systemic cardiovascular function. However, the role of extra‐hypothalamic and thalamic dopaminergic (DA) inputs in this circuit remains underexplored. Using retrograde neuroanatomical tracing and high contrast confocal imaging methods, we investigated the projections and morphology of the discrete thalamic DA neuron groups in the dorsal hypothalamic area and their contribution to the PVN‐RVLM neural circuit. We found that DA neuron subgroups in the Zona Incerta (Zi; 60%) and Reuniens thalamic nuclei (Re; 40%) were labeled comparably to the PVN (85%) after a retrograde tracer was injected into the RVLM/C1 (P < 0.01 mean ± SEM). The Re/Zi DA neuron subgroups were characterized by angulated cell bodies, superiomedial and inferiomedial projections reaching the contralateral Re/Zi and ipsilateral PVN DA neurons respectively. Ultimately, we deduced that the DA projections of the Re/Zi to the PVN contribute to the PVN‐RVLM/C1 neural circuit. As a result of these connections, the Re/Zi DA neuron groups may regulate preautonomic sympathetic events associated with the PVN‐RVLM pathway. Anat Rec, 300:1307–1314, 2017.