Anomalous expression of chloride transporters in the sclerosed hippocampus of mesial temporal lobe epilepsy patients

The Na+-K+-Cl-cotransporter 1 and K+-Cl-cotransporter 2 regulate the levels of intracellular chloride in hippocampal cells.Impaired chloride transport by these proteins is thought to be involved in the pathophysiological mechanisms of mesial temporal lobe epilepsy.Imbalance in the relative expression of these two proteins can lead to a collapse of Cl-homeostasis,resulting in a loss of gamma-aminobutyric acid-ergic inhibition and even epileptiform discharges.In this study,we investigated the expression of Na+-K+-Cl-cotransporter 1 and K+-Cl-cotransporter 2 in the sclerosed hippocampus of patients with mesial temporal lobe epilepsy,using western blot analysis and immunohistochemistry.Compared with the histologically normal hippocampus,the sclerosed hippocampus showed increased Na+-K+-Cl-cotransporter 1 expression and decreased K+-Cl-cotransporter 2 expression,especially in CA2 and the dentate gyrus.The change was more prominent for the Na+-K+-Cl-cotransporter 1 than for the K+-Cl-cotransporter 2.These experimental findings indicate that the balance between intracellular and extracellular chloride may be disturbed in hippocampal sclerosis,contributing to the hyperexcitability underlying epileptic seizures.Changes in Na+-K+-Cl-cotransporter 1 expression seems to be the main contributor.Our study may shed new light on possible therapies for patients with mesial temporal lobe epilepsy with hippocampal sclerosis.     

Protective effects of edaravone on white matter pathology in a novel mouse model of Alzheimer’s disease with chronic cerebral hypoperfusion

White matter lesions (WMLs) caused by cerebral chronic hypoperfusion (CCH) may contribute to the pathophysiology of Alzheimer’s disease (AD). However, the underlying mechanisms and therapeutic approaches have yet to be totally identified. In the present study, we investigated a potential therapeutic effect of the free radical scavenger edaravone (EDA) on WMLs in our previously reported novel mouse model of AD (APP23) plus CCH with motor and cognitive deficits. Relative to AD with CCH mice at 12 months (M) of age, EDA strongly improved CCH-induced WMLs in the corpus callosum of APP23 mice at 12 M by improving the disruption of white matter integrity, enhancing the proliferation of oligodendrocyte progenitor cells, attenuating endothelium/astrocyte unit dysfunction, and reducing neuroinflammation and oxidative stress. The present study demonstrates that the long-term administration of EDA may provide a promising therapeutic approach for WMLs in AD plus CCH disease with cognitive deficits.     

The search for NKCC1-selective drugs for the treatment of epilepsy: Structure–function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A

The Na⁺–K⁺–Cl⁻ cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl⁻ concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~ 5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure–function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC₅₀s) of bumetanide were 0.68 (hNKCC1A) and 4.0 μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC₅₀s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.     

MCP-1-mediated activation of microglia promotes white matter lesions and cognitive deficits by chronic cerebral hypoperfusion in mice

Microglia activation played a vital role in the pathogenesis of white matter lesions (WMLs) by chronic cerebral hypoperfusion. In addition, hypoxia induced up-regulated expression of MCP-1, promotes the activation of microglia. However, the role of MCP-1-mediated microglia activation in chronic cerebral ischemia is still unknown. To explore that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in mice. The activation of microglia and the related signal pathway p38MAPK/PKC in white matter, and working memory of mice were observed. We found that stenosis of common carotid arteries could induce MCP-1-mediated activation of microglia through p38MAPK/PKC pathway and white matter lesions. Taken together, our findings represent a novel mechanism of MCP-1 involved in activation of microglia and provide a novel therapeutical strategy for chronic cerebral hypoperfusion.     

Astrocyte ERK phosphorylation precedes K+‐induced swelling but follows hypotonicity‐induced swelling

Hypotonicity following water intoxication and/or salt loss leads to mainly astrocytic brain swelling. Astrocytic swelling also occurs following brain trauma or ischemia, together with an increase in extracellular K⁺ ([K⁺]_o), stimulating a bumetanide/furosemide/ethacrynic acid‐inhibitable cotransporter, NKCC1, that accumulates Na⁺ and K⁺ together with 2 Cl^‐ and osmotically obliged water. Either type of swelling may become fatal and is associated with phosphorylation of extracellular regulated kinases 1 and 2 (ERK_1/2). Only the swelling associated with elevated [K⁺]_o, leads to an increase in astrocytic proliferation and in expression of the astrocytic marker, glial fibrillary acidic protein. These differences prompted us to investigate key aspects of the molecular pathways between hypotonicity‐induced and high‐K⁺‐mediated swelling in primary cultures of mouse astrocytes. In the latter Ca²⁺‐mediated, AG1478‐inhibitable transactivation of the epidermal growth factor (EGF) receptor leads, via bumetanide‐inhibitable activation of the mitogen activated protein (MAP) kinase pathway to ERK phosphorylation and to NKCC1‐mediated swelling. In the former, inhibition of the MAP kinase pathway, but not of EGF receptor activation, abolishes ERK phosphorylation, but has no effect on swelling, indicating that activation of ERK is a result, not a cause, of the swelling.     

L-carnitine enhances axonal plasticity and improves white-matter lesions after chronic hypoperfusion in rat brain

Chronic cerebral hypoperfusion causes white-matter lesions (WMLs) with oxidative stress and cognitive impairment. However, the biologic mechanisms that regulate axonal plasticity under chronic cerebral hypoperfusion have not been fully investigated. Here, we investigated whether L-carnitine, an antioxidant agent, enhances axonal plasticity and oligodendrocyte expression, and explored the signaling pathways that mediate axonal plasticity in a rat chronic hypoperfusion model. Adult male Wistar rats subjected to ligation of the bilateral common carotid arteries (LBCCA) were treated with or without L-carnitine. L-carnitine-treated rats exhibited significantly reduced escape latency in the Morris water maze task at 28 days after chronic hypoperfusion. Western blot analysis indicated that L-carnitine increased levels of phosphorylated high-molecular weight neurofilament (pNFH), concurrent with a reduction in phosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN), and increased phosphorylated Akt and mammalian target of rapamycin (mTOR) at 28 days after chronic hypoperfusion. L-carnitine reduced lipid peroxidation and oxidative DNA damage, and enhanced oligodendrocyte marker expression and myelin sheath thickness after chronic hypoperfusion. L-carnitine regulates the PTEN/Akt/mTOR signaling pathway, and enhances axonal plasticity while concurrently ameliorating oxidative stress and increasing oligodendrocyte myelination of axons, thereby improving WMLs and cognitive impairment in a rat chronic hypoperfusion model.     

Bumetanide inhibits rapid kindling in neonatal rats

Purpose: To examine the effects of bumetanide, a selective blocker of Na⁺‐K⁺‐2Cl^? cotransporter (NKCC1), on hippocampal excitability and rapid kindling in immature rats. Methods: Studies were performed in Wistar rats of three ages: postnatal day 11 (P11, neonatal), P14 (postneonatal), and P21 (preadolescent). Bumetanide (0.2, 0.5, 2.5 mg/kg) was given intraperitoneally 20 min prior to the beginning of the studies. Hippocampal excitability was examined by measuring threshold and duration of afterdischarge, which had been elicited by electrical stimulation of ventral hippocampus. Kindling procedure consisted of 80 electrical stimulations of ventral hippocampus, delivered every 5 min. Results: At P11, bumetanide (0.5 mg/kg) increased the baseline hippocampal afterdischarge threshold and shortened the afterdischarge duration. Bumetanide delayed the occurrence, and reduced the number of full motor seizures during kindling, and prevented the development of kindling‐induced enhanced seizure susceptibility in a majority of animals. At P14, bumetanide (0.5 mg/kg) induced no significant antiepileptic effects, although suppression of hippocampal excitability and inhibition of kindling were observed in a subset of animals. At P21, bumetanide (0.2; 2.5 mg/kg) exerted no effects on hippocampal excitability and kindling progression. Discussion: The obtained results provide further evidence that bumetanide may be beneficial for treating neonatal seizures, and that NKCC1 represents a potential target for antiepileptic interventions in the immature brain.     

Phosphodiesterase III inhibition promotes differentiation and survival of oligodendrocyte progenitors and enhances regeneration of ischemic white matter lesions in the adult mammalian brain

Vascular dementia is caused by blockage of blood supply to the brain, which causes ischemia and subsequent lesions primarily in the white matter, a key characteristic of the disease. In this study, we used a chronic cerebral hypoperfusion rat model to show that the regeneration of white matter damaged by hypoperfusion is enhanced by inhibiting phosphodiesterase III. A rat model of chronic cerebral hypoperfusion was prepared by bilateral common carotid artery ligation. Performance at the Morris water-maze task, immunohistochemistry for bromodeoxyuridine, as well as serial neuronal and glial markers were analyzed until 28 days after hypoperfusion. There was a significant increase in the number of oligodendrocyte progenitor cells in the brains of patients with vascular dementia as well as in rats with cerebral hypoperfusion. The oligodendrocyte progenitor cells subsequently underwent cell death and the number of oligodendrocytes decreased. In the rat model, treatment with a phosphodiesterase III inhibitor prevented cell death, markedly increased the mature oligodendrocytes, and promoted restoration of white matter and recovery of cognitive decline. These effects were cancelled by using protein kinase A/C inhibitor in the phosphodiesterase III inhibitor group. The results of our study indicate that the mammalian brain white matter tissue has the capacity to regenerate after ischemic injury.     

Age- and region-specific effects of anticonvulsants and bumetanide on 4-aminopyridine-induced seizure-like events in immature rat hippocampal-entorhinal cortex slices

Purpose: Seizure‐like events (SLEs) induced by 4‐aminopyridine in rat organotypic slices cultures, which are prepared early after birth, are resistant to standard antiepileptic drugs. In this study we tested the hypothesis that pharmacoresistance may be an intrinsic property of the immature brain. Methods: Frequently recurring SLEs presumably representing status epilepticus were induced by 4‐aminopyridine in acute rat hippocampal–entorhinal cortex slices obtained from postnatal day 3–19 (P3–P19), and the effects of carbamazepine, phenytoin, valproic acid, and phenobarbital were examined. In addition, bumetanide was tested, which blocks the Na⁺‐K⁺‐2Cl⁻ (NKCC1) cotransporter, and also acetazolamide, which blocks the carbonic anhydrase and thereby the accumulation of bicarbonate inside neurons. Results: The efficacy of all antiepileptic drugs in blocking SLEs was dependent on postnatal age, with low efficacy in P3–P5 slices. Antiepileptic drugs suppressed SLEs more readily in the medial entorhinal cortex (ECm) than in the CA3. In P3–P5 slices, valproic acid and phenobarbital increased both tonic and clonic seizure‐like activities in the CA3, whereas phenytoin and carbamazepine blocked tonic‐like but prolonged clonic‐like activity. In P3–P5 slices, bumetanide often blocked SLEs in the CA3, but was not as effective in the ECm. Like with other antiepileptic drugs, the seizure‐suppressing effects of acetazolamide increased with postnatal age. Conclusion: We conclude that pharmacoresistance may be inherent to very immature tissue and suggest that expression of the NKCC1 cotransporter might contribute to pharmacoresistance.     

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1‐adrenergic Na+/K+‐ATPase stimulation and glycogenolysis

The cotransporter of Na⁺, K⁺, 2Cl^–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K⁺ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na⁺/K⁺‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na⁺, K⁺, and Cl^– × Cl^–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na⁺/K⁺‐ATPase activity is stimulated by elevated K⁺ or the β‐adrenergic agonist isoproterenol. Extracellular K⁺ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β₁‐adrenergic antagonist betaxolol, but not by ICI118551, a β₂‐adrenergic antagonist. A large part of the β₁‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na⁺/K⁺‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot. © 2014 Wiley Periodicals, Inc.     

Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brains angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.     

Na(+)-dependent chloride transporter (NKCC1)-null mice exhibit less gray and white matter damage after focal cerebral ischemia.

We previously demonstrated that pharmacological inhibition of Na(+)-K(+)-Cl- cotransporter isoform 1 (NKCC1) is neuroprotective in in vivo and in vitro ischemic models. In this study, we investigated whether genetic ablation of NKCC1 provides neuroprotection after ischemia. Focal ischemia was induced by 2 hours occlusion of the left middle cerebral artery (MCAO) followed by 10 or 24 hours reperfusion. Two hours MCAO and ten or twenty-four hours reperfusion caused infarction (approximately 85 mm3) in NKCC1 wild-type (NKCC1(+/+)) mice. Infarction volume in NKCC1(-/-) mice was reduced by approximately 30% to 46%. Heterozygous mutant (NKCC1(+/-)) mice showed approximately 28% reduction in infarction (P>0.05). Two hours MCAO and twenty-four hours reperfusion led to a significant increase in brain edema in NKCC1(+/+) mice. In contrast, NKCC1(+/-) and NKCC1(-/-) mice exhibited approximately 50% less edema (P<0.05). Moreover, white matter damage was assessed by immunostaining of amyloid precursor protein (APP). An increase in APP was detected in NKCC1(+/+) mice after 2 hours MCAO and 10 hours reperfusion. However, NKCC1(-/-) mice exhibited significantly less APP accumulation (P<0.05). Oxygen-glucose deprivation (OGD) induced approximately 67% cell death and a fourfold increase in Na+ accumulation in cultured NKCC1(+/+) cortical neurons. OGD-mediated cell death and Na+ influx were significantly reduced in NKCC1(-/-) neurons (P<0.05). In addition, inhibition of NKCC1 by bumetanide resulted in similar protection in NKCC1(+/+) neurons and astrocytes (P<0.05). These results imply that stimulation of NKCC1 activity is important in ischemic neuronal damage.     

Both NKCC1 and anion exchangers contribute to Cl⁻ accumulation in postnatal forebrain neuronal progenitors

Neuronal progenitors are continuously generated in the postnatal rodent subventricular zone and migrate along the rostral migratory stream to supply interneurons in the olfactory bulb. Nonsynaptic GABAergic signaling affects the postnatal neurogenesis by depolarizing neuronal progenitors, which depends on an elevated intracellular Cl^? concentration. However, the molecular mechanism responsible for Cl^? accumulation in these cells still remains elusive. Using confocal Ca²⁺ imaging, we found that GABA depolarization‐induced Ca²⁺ increase was either abolished by bumetanide, a specific inhibitor of the Na⁺–K⁺–2Cl^? cotransporter, or reduced by partial replacement of extracellular Na⁺ with Li⁺, in the HEPES buffer but not in the CO₂/ buffer. GABA depolarization‐induced Ca²⁺ increase in CO₂/ buffer was abolished by a combination of bumetanide with the anion exchanger inhibitor DIDS or with the carbonic anhydrase inhibitor acetozalimide. Using gramicidin‐perforated patch‐clamp recording, we further confirmed that bumetanide, together with DIDS or acetozalimide, reduced the intracellular chloride concentration in the neuronal progenitors. In addition, with BrdU labeling, we demonstrated that blocking of the Na⁺–K⁺–2Cl^? cotransporter, but not anion exchangers, reduced the proliferation of neuronal progenitors. Our results indicate that both the Na⁺–K⁺–2Cl^? cotransporter and anion exchangers contribute to the elevated intracellular chloride responsible for the depolarizing action of GABA in the postnatal forebrain neuronal progenitors. However, the Na⁺–K⁺–2Cl^? cotransporter displays an additional effect on neuronal progenitor proliferation.     

Na+–K+–2Cl− Cotransport Inhibitor Attenuates Cerebral Edema Following Experimental Stroke via the Perivascular Pool of Aquaporin-4

Introduction  

The Na⁺–K⁺–2Cl⁻ cotransporter localized in the brain vascular endothelium has been shown to be important in the evolution of cerebral edema following experimental stroke. Previous in vivo studies have demonstrated that bumetanide, a selective Na⁺–K⁺–2Cl⁻ cotransport inhibitor, attenuates ischemia-evoked cerebral edema. Recently, bumetanide has been shown to also inhibit water permeability via aquaporin-4 (AQP4) expressed in Xenopus laevis oocytes. We tested the hypothesis that the perivascular pool of AQP4 plays a significant role in the anti-edema effect of bumetanide by utilizing wild-type (WT) mice as well as mice with targeted disruption of α-syntrophin (α-Syn^−/−) that lack the perivascular pool of AQP4.     

Inhibition of Na(+)-K(+)-Cl(-) cotransporter during focal cerebral ischemia decreases edema and neuronal damage

Our previous study demonstrated that pharmacological inhibition of the Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) during ischemia and reperfusion attenuated neuronal damage and edema. In this study, we further investigated whether NKCC1 activity contributes to ischemic damage during either ischemia or reperfusion. Immunoblotting revealed that expression of NKCC1 protein was increased following 2-h focal ischemia in cerebral cortex. A sustained up-regulation of NKCC1 in cortex was detected at 4, 8, 12, and 24 h of reperfusion. An increase in the phosphorylated NKCC1 (NKCC1-p) was found at 4 and 8 h of reperfusion. In striatum, a significant increase in NKCC1 expression occurred between 4 and 24 h of reperfusion and no elevation of NKCC1-p signal was observed. Artificial cerebral spinal fluid (aCSF) or 100 microM bumetanide in aCSF were continuously microdialyzed into left cortices either 1 h prior to ischemia plus 2-h ischemia, or only during 24-h reperfusion. Infarction volume was significantly decreased in the pre-ischemic bumetanide-treated group (P<0.05) but not in the post-ischemic treatment group (P>0.05). In addition, pre-ischemic bumetanide treatment reduced the ipsilateral water content increase by 70% (P<0.05). Inhibition of NKCC1 did not attenuate poly (ADP-ribose) polymerase cleavage or the number of TUNEL-labeled apoptotic cells in ischemic brains. These results suggest that inhibition of NKCC1 attenuates cytotoxic edema and necrotic neuronal death during focal ischemia. Activation of NKCC1 activity plays a role in the early stage of ischemic damage.     

Contributions of the Na+/K+‐ATPase,NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

Network activity in the brain is associated with a transient increase in extracellular K⁺ concentration. The excess K⁺ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na⁺/K⁺/2Cl^? cotransporter 1 (NKCC1), and/or Na⁺/K⁺‐ATPase activity. Their individual contribution to [K⁺]_o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na⁺/K⁺‐ATPase and to resolve their involvement in clearance of extracellular K⁺ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K⁺]_o increases above basal levels. Increased [K⁺]_o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K⁺ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K⁺ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K⁺]_o increase. In contrast, inhibition of the different isoforms of Na⁺/K⁺‐ATPase reduced post‐stimulus clearance of K⁺ transients. The astrocyte‐characteristic α2β2 subunit composition of Na⁺/K⁺‐ATPase, when expressed in Xenopus oocytes, displayed a K⁺ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K⁺]_o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na⁺/K⁺‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K⁺ recovery in native hippocampal tissue while Kir4.1 and Na⁺/K⁺‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622     

Microbial lipopolysaccharide-induced inflammation contributes to cognitive impairment and white matter lesion progression in diet-induced obese mice with chronic cerebral hypoperfusion

Aims

White matter lesions (WMLs) are involved in the pathological processes leading to cognitive decline and dementia. We examined the mechanisms underlying the exacerbation of ischemia-induced cognitive impairment and WMLs by diet-induced obesity, including lipopolysaccharide (LPS)-triggered neuroinflammation via toll-like receptor (TLR) 4.

Methods

Wild-type (WT) and TLR4-knockout (KO) C57BL/6 mice were fed a high-fat diet (HFD) or low-fat diet (LFD), and subjected to bilateral carotid artery stenosis (BCAS). Diet groups were compared for changes in gut microbiota, intestinal permeability, systemic inflammation, neuroinflammation, WML severity, and cognitive dysfunction.

Results

In WT mice, HFD induced obesity and increased cognitive impairment and WML severity compared with LFD-fed mice following BCAS. HFD caused gut dysbiosis and increased intestinal permeability, and plasma LPS and pro-inflammatory cytokine concentrations. Furthermore, HFD-fed mice had higher LPS levels and higher neuroinflammatory status, including increased TLR4 expression, in WMLs. In TLR4-KO mice, HFD also caused obesity and gut dysbiosis but did not increase cognitive impairment or WML severity after BCAS. No difference was found between HFD- and LFD-fed KO mice for LPS levels or inflammatory status in either plasma or WMLs.

Conclusion

Inflammation triggered by LPS–TLR4 signaling may mediate obesity-associated exacerbation of cognitive impairment and WMLs from brain ischemia.